Breeding and Biotechnology of Plant and Flower
F. Keykha Akhar; A.R. Bagheri; N. Moshtaghi; M. Fakhrfeshani
Abstract
Introduction
Flower color is one of the most significant characteristics in ornamental plant breeding. New varieties of various plants in relation to their flower color have been obtained by monitoring the expression levels of genes involved or regulating the flavonoid and anthocyanin biosynthesis ...
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Introduction
Flower color is one of the most significant characteristics in ornamental plant breeding. New varieties of various plants in relation to their flower color have been obtained by monitoring the expression levels of genes involved or regulating the flavonoid and anthocyanin biosynthesis pathway. Flavonoids possess significant and diverse biological functions. They are the major pigments for flowers, fruits, seeds, and leaves. They are natural products that contain a C6-C3-C6 carbon framework and are synthesized by a branched pathway that yields both colored and colorless compounds. The gene encoding chalcone isomerase (CHI) is among the genes and enzymes identified in the flavonoid pathway. This enzyme catalyzes the isomerization of naringenin chalcone into the corresponding flavanone. CHI enzyme belongs to the family of isomerases, specifically the class of intramolecular lyases. Chalcone isomerase has a core 2-layer alpha/beta structure and has attracted much attention recently due to its role in stress response and pigment production. One of the most effective methods of genetic engineering is the reduction of flower pigments by suppression of required enzymes for their biosynthesis. RNA interference (RNAi) has provided the tool for the investigation of genes involved in the production of flower color. Silencing of any gene in the anthocyanin biosynthetic pathway can result in reduced or inhibited anthocyanin production. RNAi technology is an effective gene silencing method and a powerful tool for studying gene function and development of new traits by transformation of viral RNA or hairpin RNA (hpRNA) constructs into plants. The processing of dsRNA into 21-23-nt small interfering RNAs (siRNAs), and the mediators of RNAi, triggers cognate mRNA degradation. The hpRNAi methodology simply requires a transgene construct containing an inversely-repeated sequence of the target gene flanked with a promoter and terminator which effectively function in plants.
Material and Methods
In this research, with the design and construction of chiRNAi, the transformation of the RNAi construct was carried out of Petunia plants. Potted plants of P. hybrida were grown under standard greenhouse conditions (16-17°C night temperature and 21-24°C day temperature and photoperiod 16/8 (light/dark)). The RNAi construct including the 530 bp cds of the chalcone isomerase (chi) gene and 741 bp of pdk gene as intron between chi sense and antisense were used for transient RNAi-induced silencing. The pBI121-chi530 plasmids were introduced into A. tumefaciens strain LBA4404 by electroporation method. Colonies of A. tumefaciens carrying the desired plasmid were screened by PCR with specific primers for chi gene. RNAi construct co-cultured with petunia’s leave. Samples was kept in dark condition for 3 days and then transferred to branch induction media. Samples were investigated for phenotypical changes and chi gene expression by qRT-PCR.
Results and Discussion
Transgenic lines showed a reduced number of pigments and a faded flower color. So that, in purple petunia, was shown 5 phenotypical groups. These groups was indicated different levels of chi gene silencing. In pink petunia was seen two groups of phenotypical changes. In these plants, chi-RNAi construct was reduced pigment production and so, these plants had faded colors in petals. Also, the chi gene expression was reduced in all transgenic lines. Generally, the results of this research showed that RNAi can be used as an efficient method for gene silencing. The application of gene silencing can indicate the gene’s function in biosynthesis pathways of various components such as anthocyanins. In addition, the chalcone isomerase gene was identified as one of the effective genes in anthocyanin biosynthesis pathway in Petunia plants that could be involved in the production of color in these plants; hence, chi gene silencing resulted in clear phenotypic alterations in this plant.
Conclusion
In general the concentration of the target mRNA in a particular tissue could be a factor that influences silencing efficiency. At very low levels of gene expression, small amounts of the silencing target, mRNA, could be completely degraded by the RNA-induced silencing complex (RISC), whereas the presence of higher amounts of the target mRNA may result in incomplete silencing, allowing some residual functional mRNA to be translated into the corresponding protein. This research demonstrated the hpRNA construct has been successfully established for floral tissues of P. hybrida. The hpRNA construct was developed for chi-RNAi silencing of one of the key genes in the anthocyanin biosynthetic pathway in Petunia flowers. The silencing of the chi gene is a prototype for the modification of the anthocyanin biosynthetic pathway in Petunia through gene suppression. This strategy could also be useful for rapid functional analysis of other genes involved in flower development.
ahmad sharifi; fatemeh keykha; mahboobeh yazdi; abdolreza bagheri
Abstract
Introduction: Lily (Lilium spp.) is a genus of herbaceous flowering plants, which consisted of many beautiful ornamental species with large prominent flowers. Most species are native to the Northern hemisphere temperate, though their range extends into the Northern subtropics. Some specific hybrids of ...
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Introduction: Lily (Lilium spp.) is a genus of herbaceous flowering plants, which consisted of many beautiful ornamental species with large prominent flowers. Most species are native to the Northern hemisphere temperate, though their range extends into the Northern subtropics. Some specific hybrids of Lilium spp. have been developed as cut flower in controlled conditions and in some cases can be grown as pot plant. Propagation rate of lily in natural clonal propagation methods is very low and one year produces of 1-2 bulblets per bulb scale. There is also possibility of disease transmission; so that, tissue culture techniques has provided an efficient method for its micropropagation.
Materials and Methods: In this study, two separate experiments under In vitro conditions the bulblet regeneration from thin cell layer (TCL) explants of Lilium spp. was investigated. In the first experiment, after two months the effect of TCL explants with 1, 3 and 5 mm thickness on MS medium containing 1 mg/l BA, 2ip and kin in combination with 0.5 mg/l NAA on regeneration parameters were assayed. In the second experiment to determine the effect of cultivar and cytokinin types, 3mm thickness TCL explants of five cultivars (Robina, Donato, Nymph, Lessoto and Roxana) were tested on MS medium containing different plant growth regulator (PGR) compounds including BA, kin, 2ip and TDZ at concentration of 1 mg/l in combination with 0.5 mg/l NAA. The regeneration parameters were assayed after four months. In all experiments, the medium was adjusted to pH 5.8 and autoclaved at 121°C for 15 min. All cultures were incubated at 25 ± 2°C with a 16 h photoperiod under cool white flourescent lights (30 µmol/m2).
Results and Discussion: According to the first experiment results, plant growth regulator of BA in all of surveyed parameters except root number was better than other PGRs and explants with 3 mm thickness was the best in all of parameters. The interaction of PGR and explants was significant, however maximum bulblet regeneration was observed in TCL explants with 3 mm thickness in all of PGR treatments (100%). While 1 mm thickness TCL in 2ip and 1 and 5 mm thickness TCL in Kin had the least regeneration percentage. Results revealed that the interaction of explants and medium is a key factor for suitable establishment, regeneration and growth of TCLs. Bulb dormancy is one of the limiting factors in regeneration of bulbous crop species. It seems under In vitro condition explants size and PGR combination of media especially cytokinin affected on breaking of dormancy. Maximum number of leaves and dry weight of bulblets in medium containing BA was significantly higher compared with other treatments. Most of studies confirmed the positive effect of BA on regeneration of lily. The function of cytokinin in plant promoted cell division and differentiation, which lead to growth and maintaining cells in meristematic status.
Result of second experiment showed that cultivar was one of the effective factors on regeneration trait. Oriental lily cultivar "Roxana" had the highest number of roots, bulblets, dry weight and length of plantlets and "Nymph" cultivar showed the lowest percentage of regeneration, dry weight, length of plantlets and rooting obtained. In all of cultivars BA induced more organogenesis percentage and plantlet dry weight, while TDZ induced more rooting percentage.The interaction of cultivar and PGR treatments on percentage of regenerated bulblets and rooting were significant. "Nymph" cultivar had minimum percentage of regeneration and rooting in medium containing TDZ and Kin. Furthermore, "Roxana" cultivar in medium containing BA showed the best dry weight comparison to other treatments.
Conclusion Lily has widely used in the floral industry as a cut flower or potted plant. In recent years, tissue culture was developed as reliable and highly effective method to overcome its limitations of vegetative propagation. The most advantage of this method is high multiplication rate and disease free propagation. In this study, bulblet regeneration of lilium Spp. from TCL explants under in vitro condition was considered as a highly efficient procedure for its micropropagation. With optimization of TCL system some parameters such as exogenously applied plant growth regulators, cultivar, explants types were investigated. Favorable conditions for bulblet regeneration were achieved with 3 mm thickness TCLs in MS medium containing 1 mg/l BA with 0.5 mg/l NAA. This protocol can be used for rapid micropropagation of many cultivars.
ahmad sharifi; Mahdiyeh Kharrazi; Fatemeh Keykha Akhar; Abdolreza Bagheri; Elahe Samari; Maryam Moradiyan
Abstract
Introduction: Gerbera is one of the most important ornamental plants in the world. The importance of Gerbera is due to its beauty, diversity and economically aspects. Traditional propagation methods such as crown division and cutting methods are not suitable for obtaining disease free plants and rapid ...
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Introduction: Gerbera is one of the most important ornamental plants in the world. The importance of Gerbera is due to its beauty, diversity and economically aspects. Traditional propagation methods such as crown division and cutting methods are not suitable for obtaining disease free plants and rapid multiplication. These methods also do not have the capacity to fulfill global demands. Therefore, obtaining efficient protocol for micropropagation of this ornamental plant is necessary.
Materials and Methods: In this study the effect of various factors on in vitro regeneration, proliferation, rooting and acclimation of gerbera capitulum explants were analyzed in four separate experiments. Capitulum explants were first washed with running tap water for 30 min then surface sterilized by dipping in 1.5% sodium hypochlorite solution for 15 min and rinsed with sterile distilled water, followed by immersing in 0.1 % mercuric chloride solution for 10 min. To remove mercuric chloride residue, capitulum was rinsed with sterile distilled water. Subsequent washing was done with sterile distilled water for three times. Sterilization steps were done under laminar air flow hood. For regeneration, eight genotypes of gerbera capitulum explants (Famous, Sunway, Red Pearl, Pink Snow, Popov, Balance, Dune, Eagle)were cultured on solid MS medium containing several cytokynins, BA, TDZ, 2IP or KIN (4 mg/l) in combination with IAA (0.2 mg/l). In proliferation stage, the effect of different concentrations of BA was evaluated on proliferation rate of Sunway regenerated explants. In the rooting stage, Sunway genotype plantlets were cultured on ½ MS medium containing NAA, IBA or IAA (1 mg/l) or ½ MS medium without any hormones. The pH of the medium was adjusted to 5.7-5.8 prior to autoclaving (15 min at 121 oC and 1.5 kg.cm-2 pressure). The cultures were incubated in a growth chamber at 25±2 oC with a 16-h photoperiod (2500-3000 Lux) provided by cool-white fluorescent lamps. For acclimation of rooted plantlets, different substrates used as follow: 1- perlite, 2- perlite: Cocopeat, 3- Cocopeat: peat moss, 4- Cocopeat: peat moss; treated with fungicide.
After 30 days, the response of explants was evaluated for each experiment. Data preparation was done in the Excel program and data analysis was done using JMP-8 software. Mean comparison of the treatments was done by Tukey test and finally the charts were drawn using the Excel program.
Results and Discussion:The results of regeneration stage showed that application of MS media containing kinetin or 2IP did not make an appropriate response to capitulum explants and no regeneration was observed in this condition. The medium containing 4 mg/l BA and 0.2 mg/l IAA indicated the highest percentage of regeneration in all genotypes.
The highest regeneration was observed in Sunway genotype with an average of 21.96%. On the other hand no regeneration was observed in Eagle genotype. In terms of the number of regenerated plantlet, the highest number (61.2) was attributed to the Sunway genotype while no plantlet was recorded for Eagle genotype. No significant differences were also observed between Pink Snow and Dune genotypes.
For the proliferation stage, only Sunway genotype was utilized due to its vigorous growth in comparison to other genotypes. In this stage, the highest (6 regenerated plantlets) and the lowest (1 regenerated plantlet) regeneration rate were observed in MS medium containing 2 mg/l BA and hormone-free medium, respectively. Hormone-free ½ MS medium and ½ MS medium containing 1 mg/l IAA or IBA, indicated the highest rooting rate (100% rooting) while medium containing 1 mg/l NAA showed 55% rooting rate. It seems that the application of NAA in the medium composition had the lowestimpact on the rooting of regenerated plantlets. At the end of the experiment, the highest (90.42%) and the lowest (47.5%) acclimation rate was obtained in peat moss + cocopeat + fungicide medium and perlite medium, respectively.
Conclusions: Generally, for shoot induction of gerbera through capitulum culture, application of MS medium containing 4 mg/l BA and 0.2 mg/l IAA is recommended. It is also concluded that for proliferation stage, the MS medium containing 2 mg/l BA showed the highest rate of regeneration. Using of Hormone-free ½ MS medium is economically affordable. Finally for acclimation of the plantlets, application of peat moss + cocopeat + fungicide medium is recommended.
A. Nezami; Seyed Mmohammad javad Mousavi; S. Nezami; E. Izadi Darbandi; M. Yousef Sani; fatemeh keykha akhar
Abstract
Calendula (Calendula officinalis) is relatively cold tolerant plant, but in some years plant seriously injured due to harsh winter. In order to evaluate freezing tolerance of calendula an experiment was carried out at college of Agriculture, Ferdowsi University of Mashhadin a factorial-completely randomized ...
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Calendula (Calendula officinalis) is relatively cold tolerant plant, but in some years plant seriously injured due to harsh winter. In order to evaluate freezing tolerance of calendula an experiment was carried out at college of Agriculture, Ferdowsi University of Mashhadin a factorial-completely randomized design with three replications and plants of two sowing dates (summer and autumn) were exposed to12 temperatures (0, -2, -4, -6, -8, -10, -12, 14- ,16-, -18, -20 and -22oC). Seeds of calendula plants were sown in summer (summer plant) and autumn (autumn plant) in the bed and in six to eight-leaf stages were transplanted to the pots. After the cold acclimation in natural condition, freezing stress was applied with using a thermo gradient freezer. To employ stability of cytoplasmic membrane, percentage of electrolyte leakage (EL%) was measured after freezing. Also survival percentage (Su%) and regrowth of calendula plants determined after three weeks recovery. Leaves EL% in autumn plants was significantly more than summer plants and autumn plants have higher Su%, but plant height, number of lateral branches, numbers of reproductive traits, total dry matter, vegetative and reproductive dry matter in summer plants were more than autumn plants. However, there were no difference between calendula plants for LT50el in both autumn and summer plants, but there was significant difference between them for LT50su and total dry matter, and LT50su and reduced dry matter temperature50 (RDMT50)for summer plants were -18.6 oC and -11.3 oC and for autumn plants were -19.4 oC and -13.7 oC, respectively.
