Enhanced in vitro Shoot Regeneration and Biochemical Properties of Stevia rebaudiana using Chitosan

Stevia rebaudiana is a herbaceous perennial plant with great global demand due to its beneficial steviol glycosides(SGs) content. Current conventional breeding technique is unable to cater the need for more S. rebaudiana planting materials. Therefore, an improved in vitro shoot regeneration protocol was developed for S. rebaudiana by using chitosan. The highest fresh weight of plant (0.586 ± 0.176 g/explant), dry weight of plant (0.056 ± 0.02 g/explant) and plant height (4.94 ± 1.17 cm/explant) with maximum number of leaves (25.33 ± 6.95 /explant) were observed on explants grown in optimun treatment of MS basal medium supplemented with 1.0 mg/L of 6-benzyaminopurine (BAP) and 60 mg/L of low molecular weight (MW) chitosan after 4 weeks of culture. Scanning electron microscopy (SEM) analysis showed that new shoot primordia and shoot bud formation can be seen as early as day 3 and 5 of cultures on optimun treatment. Further biochemical assays showed that total phenolic acid content, total protein content and total hydrolyzed sugar content were recorded higher in explants cultured in optimun treatment as compared to control media. In contrast, total chlorophyll content and total flavonoids content were reduced in optimum treatment. Meanwhile, no significant difference in antioxidant activity was observed. All cultures from the optimal treatment were successfully regenerated, acclimatized and grew well with 100% survival rate. Taken together, an enhanced and efficient shoot regeneration protocol of S. rebaudiana was successfully developed which will be useful for rapid and large-scale micropropagation in future.

In vitro shoot regeneration in Myracrodruon urundeuva Fr. All

ABSTRACT Myracrodruon urundeuva Fr. All. is a tree threatened with extinction, which has wood and medicinal potential. This study aimed to analyze the in vitro shoot regeneration in M. urundeuva, in order to increase the species multiplication. Two experiments were conducted: 1) concentrations of 6-benzylaminopurine (BAP) (0.0, 2.0, 4.0, 8.0 and 16.0 µM), in association with naphthaleneacetic acid (NAA) (0.0, 1.5 and 3.0 µM), in explants (cotyledon, hypocotyl and cotyledonary node); 2) concentrations of meta-topolin (mT) (0.0, 2.0, 4.0, 8.0, 16.0 and 32.0 µM) in explants (biaxillary, medial uniaxillary and apical basal nodal segment). The percentage of explants responsive to shoot regeneration, percentage of callus explants, number of shoots and shoot length were evaluated. In the first experiment, the shoot regeneration occurred only in explants of the cotyledonary node and hypocotyl type, with the highest responsiveness percentage (76.67 %) and number of shoots (1.97 and 1.63) obtained for the cotyledonary node in the presence of 3.0 µM of NAA in association with 2.0 (1.97 shoots/explant) and 4.0 µM (1.63 shoots/explant) of mT. In the second experiment, the resolution of the obtained quadratic equation indicates that the use of basal explant with 24.59 µM of mT added to the culture medium leads to the highest number of shoots (1.86). However, despite the mT having increased the mean number of shoots, all treatments containing this cytokinin showed callus formation. As a conclusion, it is possible to regenerate shoots in M. urundeuva from the cotyledonary node using BAP in association with NAA.

Effect of plant growth regulators on in vitro culture of pineapple (Ananas comosus L. Merr) MD2 variety

Pineapples (Ananas comosus L. Merr) are fruits that belong to the Bromeliaceae family. Pineapple variety MD2 is one of the varieties that has gained a place in the market among pineapple farmers due to its high value and quality. However, it is difficult to meet the demand for planting materials using conventional propagation techniques. Hence, plant tissue culture technology is one of the methods that has been widely used in the agriculture industry that boosts up the production of pineapple planting materials within a short period and is cost-efficient. The objective of this study was to determine the effect of plant growth regulator concentration to in vitro culture of MD2 variety pineapple. In this study, the various concentrations of 6-Benzylaminopurine (BAP) and α-Naphthalene Acetic Acid (NAA) for in vitro culture of MD2 pineapple were studied. The plantlets were effectively initiated from MD2 pineapple crown on Murashige and Skoog (MS) basal salt containing 30.0 g/L sucrose, and 2.0 mg/L BAP in two months of culture. Next, the pineapple plantlet was subculture on shooting medium containing full strength solid Murashige and Skoog (MS) medium supplemented with vitamins with various concentration BAP (0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 mg/L) and NAA (0, 1.0, and 2.0 mg/L). The result obtained showed that the solid MS medium added with 30.0 g/L sucrose, without any BAP and NAA (T1) had the highest in vitro shoot regeneration. Meanwhile, the solid MS medium with 30.0 g/L sucrose with 1.0 mg/L NAA (T1) recorded the highest plantlet height (cm). The mean value for in vitro shoot regeneration in T1 and plantlet height (cm) in T1 were 2.80 (±0.5) and 4.40 (±0.3). To conclude, less amount plant hormone regulator required to obtain the mass quantity of in vitro clonal pineapple that can help solve the problem of lack of plant material in the pineapple crop industry.

Micropropagation of some Onobrychis species through in vitro shoot regeneration

Onobrychis species have an extensive spread in Turkey and adapted to various environmental conditions. In this study, it was aimed to propagate with tissue culture methods of endemic Onobrychis fallax Freyn & Sint. ex Freyn var. longifolia Aktoklu, Onobrychis stenostachya subsp. sosnowskyi, Onobrychis elata Boiss. & Balansa and worldwide commonly cultivated Onobrychis viciifolia Scop. species. For this purpose, cotyledon node explants were cultured in Murashige and Skoog (MS) media containing various concentration of 6-benzylaminopurine (BAP), thidiazuron (TDZ) or meta-Topolin (mT) alone or in combination with 0.5 mg L–1 NAA. Significant variations in number of shoots per explant was observed depending on the species and growth regulators. The greatest number of shoots per explant was obtained from 1.2 mg L–1 TDZ treatments in O. stenostachya, O. fallax and O. elata and from 0.3 mg L–1 TDZ treatments in O. viciifolia, whereby 10.58, 9.50, 5.5, 6.42 shoots were recorded. Resultant shoots were rooted in half strength MS nutrient media containing 1 or 2 mg L–1 indole-3-butyric acid (IBA). The rooting ratio was 86.11% in O. viciifolia, 50.00% in O. stenostachya and 36.11% in O. fallax and O. elata.

Application of Artificial Neural Network for Modeling and Studying In Vitro Genotype-Independent Shoot Regeneration in Wheat

Optimizing in vitro shoot regeneration conditions in wheat is one of the important steps in successful micropropagation and gene transformation. Various factors such as genotypes, explants, and phytohormones affect in vitro regeneration of wheat, hindering the ability to tailor genotype-independent protocols. Novel computational approaches such as artificial neural networks (ANNs) can facilitate modeling and predicting outcomes of tissue culture experiments and thereby reduce large experimental treatments and combinations. In this study, generalized regression neural network (GRNN) were used to model and forecast in vitro shoot regeneration outcomes of wheat on the basis of 10 factors including genotypes, explants, and different concentrations of 6-benzylaminopurine (BAP), kinetin (Kin), 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1-naphthaleneacetic acid (NAA), zeatin, and CuSO4. In addition, GRNN was linked to a genetic algorithm (GA) to identify an optimized solution for maximum shoot regeneration. Results indicated that GRNN could accurately predict the shoot regeneration frequency in the validation set with a coefficient determination of 0.78. Sensitivity analysis demonstrated that shoot regeneration frequency was more sensitive to variables in the order of 2,4-D > explant > genotype < zeatin < NAA. Results of this study suggest that GRNN-GA can be used as a tool, besides experimental approaches, to develop and optimize in vitro genotype-independent regeneration protocols.