Machine learning-mediated Passiflora caerulea callogenesis optimization.

Callogenesis is one of the most powerful biotechnological approaches for in vitro secondary metabolite production and indirect organogenesis in Passiflora caerulea. Comprehensive knowledge of callogenesis and optimized protocol can be obtained by the application of a combination of machine learning (ML) and optimization algorithms. In the present investigation, the callogenesis responses (i.e., callogenesis rate and callus fresh weight) of P. caerulea were predicted based on different types and concentrations of plant growth regulators (PGRs) (i.e., 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), 1-naphthaleneacetic acid (NAA), and indole-3-Butyric Acid (IBA)) as well as explant types (i.e., leaf, node, and internode) using multilayer perceptron (MLP). Moreover, the developed models were integrated into the genetic algorithm (GA) to optimize the concentration of PGRs and explant types for maximizing callogenesis responses. Furthermore, sensitivity analysis was conducted to assess the importance of each input variable on the callogenesis responses. The results showed that MLP had high predictive accuracy (R2 > 0.81) in both training and testing sets for modeling all studied parameters. Based on the results of the optimization process, the highest callogenesis rate (100%) would be obtained from the leaf explant cultured in the medium supplemented with 0.52 mg/L IBA plus 0.43 mg/L NAA plus 1.4 mg/L 2,4-D plus 0.2 mg/L BAP. The results of the sensitivity analysis showed the explant-dependent impact of the exogenous application of PGRs on callogenesis. Generally, the results showed that a combination of MLP and GA can display a forward-thinking aid to optimize and predict in vitro culture systems and consequentially cope with several challenges faced currently in Passiflora tissue culture.

Prediction and optimization of indirect shoot regeneration of Passiflora caerulea using machine learning and optimization algorithms.

BACKGROUND: Optimization of indirect shoot regeneration protocols is one of the key prerequisites for the development of Agrobacterium-mediated genetic transformation and/or genome editing in Passiflora caerulea. Comprehensive knowledge of indirect shoot regeneration and optimized protocol can be obtained by the application of a combination of machine learning (ML) and optimization algorithms. MATERIALS AND METHODS: In the present investigation, the indirect shoot regeneration responses (i.e., de novo shoot regeneration rate, the number of de novo shoots, and length of de novo shoots) of P. caerulea were predicted based on different types and concentrations of PGRs (i.e., TDZ, BAP, PUT, KIN, and IBA) as well as callus types (i.e., callus derived from different explants including leaf, node, and internode) using generalized regression neural network (GRNN) and random forest (RF). Moreover, the developed models were integrated into the genetic algorithm (GA) to optimize the concentration of PGRs and callus types for maximizing indirect shoot regeneration responses. Moreover, sensitivity analysis was conducted to assess the importance of each input variable on the studied parameters. RESULTS: The results showed that both algorithms (RF and GRNN) had high predictive accuracy (R2 > 0.86) in both training and testing sets for modeling all studied parameters. Based on the results of optimization process, the highest de novo shoot regeneration rate (100%) would be obtained from callus derived from nodal segments cultured in the medium supplemented with 0.77 mg/L BAP plus 2.41 mg/L PUT plus 0.06 mg/L IBA. The results of the sensitivity analysis showed the explant-dependent impact of exogenous application of PGRs on indirect de novo shoot regeneration. CONCLUSIONS: A combination of ML (GRNN and RF) and GA can display a forward-thinking aid to optimize and predict in vitro culture systems and consequentially cope with several challenges faced currently in Passiflora tissue culture.

Effect of plant growth regulators on indirect shoot organogenesis of Ficus religiosa through seedling derived petiole segments.

Ficus religiosa is known as a long-lived multipurpose forest tree. The tree plays an important role for religious, medicinal, and ornamental purposes. However, the propagation rate of Ficus religiosa is low in natural habitat so the plant tissue culture techniques are an applicable method for multiplication of this valuable medicinal plants. Thus, the aim of this study is to understand the effect of different auxin/cytokinin ratios on indirect shoot organogenesis of this plant. According to our results, the maximum callus induction frequency (100%) was obtained on Murashige and Skoog (MS) medium supplemented with 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) plus 0.05 mg/l 6-benzylaminopurine (BAP) from petiole segments. For shoot induction purpose, the yellow-brownish, friable, organogenic calli were inoculated on shoot induction medium. On MS medium supplemented with 1.5 mg/l BAP and 0.15 mg/l Indole-3-butyric acid (IBA), 96.66% of the petiole-derived calli responded with an average number of 3.56 shoots per culture. The highest root formation frequency (96.66%), root number (5.5), and root length (4.83 cm) were achieved on MS medium containing 2.0 mg/l IBA plus 0.1 mg/l Naphthaleneacetic acid (NAA). The rooted shoots were successfully transferred to field condition and the substrate with the mixture of cocopeat and perlite (1:1) had the highest survival rate (96.66%). This is the first report of an effective in vitro organogenesis protocol for F. religiosa by indirect shoot organogenesis through axenic seedling derived petiole explants, which can be efficiently employed for conservation of this important medicinal plant species as well as the utilization of active biomolecules.

The Effects of Different Media on Shoot Proliferation From the Shoot Tip of Aloe vera L.

BACKGROUND: Aloe vera L. is an important pharmaceutical plant from which several medicinal and cosmetic compounds are extracted. Aloe is naturally propagated through offset, which is a slow and expensive labor cost method with low economical income. OBJECTIVES: In this study, the effect of different media on shoot proliferation of the shoot tip of Aloe vera L. was investigated. MATERIALS AND METHODS: In vitro techniques are some of the suggested methods for rapid propagation of Aloe. In this experiment, the shoot tips of mother plants were grown in a greenhouse. After surface sterilization of the explants, they were cultured on Murashige and Skoog (1962) (MS) medium containing different concentrations of kinetin and naphthaleneacetic acid (NAA). The experiment was carried out in the form of a randomized complete design with three replications. RESULTS: The results showed that MS media containing 1.5 mg/L kinetin along with 0.15 or 0.3 mg/L NAA produced the highest percentage of proliferated shoots. In addition, the percentage of proliferated shoots in MS medium containing 2.0 or 2.5 mg/L benzylaminopurine (BAP) + 0.15 mg/L NAA was significantly higher than the other treatments. CONCLUSIONS: Analysis of the interactive effects of NAA, kinetin and BAP on shoot proliferation showed that most of the proliferated shoots produced in MS medium containing 1.0 mg/L BAP + 1.0 mg/L kinetin + 0.15 mg/L NAA were significantly different from other treatments. Rooting quality was greater in MS media containing 1.0 mg/L IBA than a 1.0 mg/L NAA treatment.

Cloning, Transformation and Expression of Human Interferon α2b Gene in Tobacco Plant (Nicotiana tabacum cv. xanthi).

BACKGROUND: Molecular farming is the production of important recombinant proteins in transgenic organisms on an agricultural scale. Interferons are proteins with antiviral and antitumor activities and can be used for viral infections and cancers treatments. OBJECTIVES: This study reports the transformation of INF α2b gene in tobacco plant for the first time in Iran. MATERIALS AND METHODS: Interferon α2b gene was amplified by PCR using specific primers containing appropriate restriction enzymes, plant highly expression sequence and Histidine tag sequence. Target sequence was cloned in plant expression vector pCAMBIA1304 and the construct named pCAMINFα. pCAMINFα was transferred to E. coli strain DH5α and plated on LB agar medium containing kanamycin 50 mgl-1. The colonies were confirmed by colony PCR and sequencing. The construct was transferred into Agrobacterium tumefaciens by freeze-thaw method and transformed colonies were confirmed by colony PCR. Tobacco plants (cultivar xanthi) were inoculated with A. tumefaciens strain LBA4404 by leaf disc method. Inoculated explants were cultured on MSII (MS + BAP 1mgl-1 + NAA 0.1 mgl-1) at 28°C and darkness for 48 hours. Then explants were transferred to selection medium containing cephotaxime (250 mgl-1) and hygromycin (15 mgl-1) in a 16/8 (day/night) h photoperiod in growth room with an irradiance of 5000 lux. Transgenic plants were regenerated and transferred to perlite. Genomic DNA was extracted from regenerated plants by Dellaporta method at 5-leaf step and transgenic lines were confirmed by PCR with specific primers. Expression of Interferon α2b gene was confirmed by dot blotting. CONCLUSIONS: Since no report of interferon alpha production in plants in Iran has been expressed yet, this research could create a field of producing this drug in tobacco, in Iran.