An Alternate Cost-effective Medium for In Vitro Regeneration of Therapeutically Important Ocimum citriodorum Vis

ABSTRACT: A novel cost-effective in vitro regeneration protocol has been evolved for the therapeutically important Ocimum citriodorum Vis. In the present study, table sugar (3%) and isabgol (Psyllium husk) (3.5%) were used as an alternate source of carbon and gelling agent respectively in Murashige and Skoog’s (MS) medium. The explant used in the current study was nodal segment. A significant observation revealed that all the cultures resulted in shoot induction and maximum number of shoots/ culture (6.04) and their average length (2.15 cm) was obtained on modified MS-medium supplemented with table sugar, isabgol and BAP. However, best root induction (95.83%) was obtained on ½ MS-medium augmented with table sugar (3%) , isabgol (3.5%) and NAA. An increase in average number of roots per shoot (6.91%) as well as average root length (2.73 cm) was also observed in the same modified medium. The in vitro regenerated plantlets were successfully transferred to the field and no notable variation was observed in their morphology. The overall cost of culture medium for in vitro propagation of O. citriodorum Vis. was reduced significantly by 92.69% when agar and sucrose were replaced by isabgol and table sugar, respectively.

Micropropagation and experimental field cultivation of Pulsatilla turczaninovii Kryl. et Serg. (Ranunculaceae)

Pulsatilla turczaninovii is an important medicinal plant, valued for high ornamental value of melliferous flowers. We assessed the efficiency of reproduction under in vitro conditions and the ex situ growth capacity of this important representative of the world flora. The seed germination percentage was assessed, followed by determination of micropropagation rate and rooting efficiency. Then, the possibility of plant development in three consecutive growing seasons was assessed. The in vitro germination percentage was approximately 55%. The highest multiplication coefficient, amounting to 5.17, was obtained on modified MS medium supplemented with 2.5 mg L−1 2iP and 1.0 mg L−1 IAA. Our study provided unique insight on biochemical background of root regeneration in P. turczaninovii. In comparison with standard auxin-supplemented rooting medium, the treatment with 1.0 mg L−1 level of ethylene precursor ACC elevated rooting by about 20%. The total content of soluble sugars was proved to be biomarker of rhizogenesis in the studied species. Their concentration was positively correlated with rooting efficiency, while a level of phenolic was positively correlated with the length of regenerated roots, and their number per rosette. The cultivation of the acclimatized material was successfully carried out and was evaluated over three subsequent years. In the third year of cultivation, the plants entered the stage of generative development and most of them bloomed profusely.

Agrobacterium-mediated Genetic Transformation of Rice var. BRRI Dhan 58

Agrobacterium mediated genetic transformation of BRRI Dhan 58 was conducted by using immature embryos following indirect regeneration. High percentage of callus induction at 96.5% was obtained when seeds of BRRI dhan 58 were cultured on modified MS medium supplemented with 2.5 mg/l 2, 4-D under dark condition. The maximum regeneration response was rerecorded when MS was supplemented with 3 mg/l BAP + 0.5 mg/l NAA and 1.0 mg/l Kn. Genetic transformation was performed using A. tumefaciens strain LBA4404 harboring pCAMBIA1301 plasmid carrying the marker genes for β-glucuronidase (GUS) and hygromycin resistance (hptII). Integration of the GUS gene into the genome of the rice plants was confirmed by PCR. The leaf segments of the PCR positive transformed plants showed the expression of GUS. The results of this study would be an effective tool for crop improvement and functional studies of gene on rice plant. Plant Tissue Cult. & Biotech. 31(1): 71-80, 2021 (June)

Propagation in Vitro and Experimental Field Cultivation ex Situ of Pulsatilla Turczaninovii Kryl. et Serg. (Ranunculaceae)

Pulsatilla turczaninovii is an important medicinal plant and a material also appreciated by gardeners for its high ornamental value. In this study we assessed the efficiency of reproduction under in vitro conditions of this important representative of the world flora. First, the germination percentage and the following plant development during three growing seasons were assessed, then we tested the possibilities of multiplication by in vitro culture methods. Our study provided new insight on biochemical background of generating adventitious roots in Pulsatilla considered difficult-to-rooting. The germination percentage of studied population reached about 55%. The highest multiplication coefficient of micro-rosettes in tissue culture, amounting to 5.17, was achieved using modified MS medium supplemented with 2.5 mg L-1 2iP and 1 mg L-1 IAA. We proved that in comparison with standard auxin-supplemented rooting medium, the treatment with addition of 1.0 mg L-1 ethylene precursor ACC elevated adventitious rooting by about 20%. The biochemical analyses revealed that total content of soluble sugars was the most impactful biomarker of adventitious rhizogenesis in studied species. Concentration of sugars was positively correlated with rooting efficiency, while the level of phenolic compounds was positively correlated with the root length and their number regenerated per single rosette.

IN VITRO RESPONSE BY Terminalia arjuna GENOTYPES DURING MICROPROPAGATION

Terminalia arjuna is an important tree of the medicinal and sericulture industry, commonly known as Arjun. It’s bark is rich in secondary metabolites makes this plant highly valuable in medicine industry to treat cardiovascular disease. Overexploitation due to high demand in medicine, low seed germination, limitations of the conventional method of propagation push this plant towards being endangered. To conserve germplasm of such tree species and meet the requirement in medicinal industry, some non-conventional propagation method like micropropagation has been developed. The present work highlighted the effect of three genotypes (G-1, G-2, and G-3) on tissue culture of T. arjuna situated at Jodhpur, Rajasthan, India. In vitro shoot proliferation was achieved on a modified MS medium enriched with BAP + additives. Among the tested genotypes, Genotype -1 showed maximum bud break response (100%) followed by G-3 (93.33 %) and G-2 (86.66%). Further multiplication of these shoots on modified MS medium containing BAP + NAA + additives gave 11.38±0.26 (G-1), 9.44±0.21 (G-2) and 10.22±0.32 (G-3) shoots. In vitro rooting was done by pulse treatment with IBA for 10 min prior to transfer on hormone free half strength MS medium containing 0.1% activated charcoal. Maximum in vitro rooting was obtained in G-1 (80%) followed by G-3 (71.11%) and G-2 (68.88%). In the present study, it was observed that optimum growth in all three genotypes required different doses of Plant Growth Regulator. Thus, by identifying and multiplying the best performing genotypes the gap between demand and supply of such medicinal plant can be fulfilled.

Cardenolide and glucosinolate accumulation in shoot cultures of Erysimum crepidifolium Rchb.

Erysimum crepidifolium Rchb. is one of the few Brassicaceae species accumulating glucosinolates as well as cardenolides. This is possibly providing a selective advantage in evolution as both compounds are part of a chemical defense system. In order to study the biosynthesis of these compounds, a regeneration protocol for E. crepidifolium using in vitro shoot cultures derived from seeds has been developed. Murashige and Skoog (MS) culture medium supplemented with various combinations of cytokinins and auxins was used. MS medium containing NAA (naphthaleneacetic acid, 0.04 mg mL−1) and BAP (6-benzylaminopurine, 0.2·10−2 mg mL−1) proved to be optimal for root formation. Plantlets developed well on modified MS medium without the use of phytohormones. About 80% of the plantlets rooted in vitro developed into intact plants after transfer to the greenhouse. Cardenolides (1.75 mg g−1 dry weight (DW)) were detected in cultured shoots on solid DDV media while glucosinolates mainly accumulated in roots where 0.025 mg g−1 FW were detected in shoots cultured on the same medium (DDV). The expression of two progesterone 5β-reductase and three Δ5-3β-hydroxysteroid dehydrogenase genes were measured in shoot cultures since the encoded enzymes are supposed to be involved in cardenolide biosynthesis. E. crepidifolium shoot cultures propagated on solid media meet the necessary requirements, i.e., clonal homogeneity, product accumulation, and gene expression, for a suitable model to study cardenolide but not glucosinolate biosynthesis.

Plantlet regeneration via nucellar embryogenesis in Citrus jambhiri

To produce true-to-type and rapid multiplication micropropagation technique was utilized to the nucellar tissue with ovular halves of C. jambhiri. Nucellar tissues were cultured in a modified MS medium supplemented with different concentrations of 2ip viz. 0.25,0.50 mg l-1 alone and in combination of 0.50 mg l-1 NAA. Initiation of cell division and differentiation of proembryogenic tissue became apparent in first 80 days.These proembryos developed into embryos through subculturing in fresh medium. Low concentration of 0.25 2ip was found more suitable for the development of embryo producing large number of fully developed embryo in comparision to the embryo produced in the high concentration of 2ip (0.50 mg l-1) and in combination of 0.25 2ip and 0.50 mg l-1 NAA. Normally developed embryos in 0.25 2ip showed best germination in the fresh medium supplemented with 0.25 mg l-1 IAA, 100 ME mg l-1 and 5mg mg l-1 amino acids within 30 days as compared to other treatments. These germinated embryos were utilized for producing disease free saplings after hardening and nurturing in laboratory conditions. The disease free saplings thus produced can be used to establish new Citrus orchards within short time.

In-Vitro Rooting Induction On The Embryo Somatic Of Dendrobium Species From Riau Province Indonesia

Rooting induction is a very important stage in in-vitro for the anticipation of the extinction, In this paper, the rooting induction of embryo somatic dendrobium species from Riau Province Indonesia is studied using different sucrose concentrations (0 g L-1; 25 g L-1; 50 g L-1; 75 g L-1), and Kinetin (0 mg L-1; 0,1 mg L-1; 1,0 mg L-1; 10 mg L-1), in a modified MS medium containing half the regular concentration of macronutrients at pH 5.6, with 50 g L-1 sucrose and 1,0 mg L-1 kinetin was optimal for a percentage of explant producing root, a number of roots, root high, root and shoot ratio (T/R Ratio).

Nitrogen assimilation in the bromeliad Ananas comosus var. ananassoides (Baker) Coppens & F.Leal grown in vitro with different sources of inorganic nitrogen

ABSTRACT Ananas comosus var. ananassoides (Baker) Coppens & F.Leal is a native ornamental bromeliad of the endangered biome Cerrado. Therefore, approaches aimed at the preservation of this species, such as in vitro cultivation and micropropagation are needed. Nitrogen (N) is absorbed by plants, mainly as NO3- and/or NH4+, and assimilated into amino acids. The aim of this work was to evaluate the N assimilation in this bromeliad. Plants were grown in vitro for seven months in modified MS medium with 15, 30, 60, and 90 mM of N as NO3-, NH4+ or NH4NO3, and then transferred to ex vitro conditions for acclimatization. Plants grown with NH4+ had high mortality. During acclimatization plants cultivated with 30, 60, and 90 mM of N as NH4NO3 showed higher biomass. With regard to N assimilation, GS and NR showed the highest activity in plants cultivated with NH4NO3, whereas plants cultivated with NH4+ had the highest GDH activity. Consequently, in vitro and ex vitro cultivation of this species with 60 mM N as NH4NO3 is recommended.