Modified MS Medium Research Articles

In vitro Rapid Regeneration of Bitter Melon (Momordica charantia L.) Using Direct Organogenesis in Kenya

Bitter melon is a traditional therapeutic vegetable in tropical and subtropical countries due to its carotenoid content, which provides health advantages. However, the production is significantly impacted by the poor seed germination rate and variability of progenies associated with the cultivation of this plant. Clonal propagation would increase the yield and usefulness of bitter melon. Momordica charantia was micropropagated using nodal explants in a modified MS medium containing vitamins (Glycine, myo-inositol, nicotinic acid pyridoxine HCl). Nodal explants were obtained from in vivo seedlings grown for 30 days in a greenhouse and then cultured on MS media with a range of BAP or KIN concentrations (0.50 - 2.5 mg/L), either alone or in combination. Shoots were induced within a minimum (6.33±0.58) day when 0.5 mg/L BAP was coupled with 0.50 mg/L KIN, resulting in the maximum shoot induction frequency (72.17±2.83%), 4.40±0.17 shoots/explant and 4.13±0.98 cm shoot length. Then, shoots were inoculated on 1/2 strength MS media either without growth regulator or with a supplement of IBA, NAA, and IAA in various concentrations (0.5 - 1.5 mg/L) to promote root induction. MS medium without growth regulator induced maximum rooting rates (93.33±2.66%) with 12.33 roots/shoot and 23.33 cm of root length. Rooted plantlets were successfully established in greenhouses after undergoing a hardening period in a regulated environment. The combination of BAP and KIN (0.50 mg/L and 0.50 mg/L) was most suitable for bitter melon shoot formation, whereas MS medium without growth regulator was optimal for rooting. This reproducible protocol for the regeneration of bitter melon plantlets would allow for rapid mass multiplication, conservation, and genetic manipulation for its agronomic improvement.

The use of tissue culture method for the study of lichenized fungi (Lobariaceae Chevall., Parmeliaceae Zenker.) of the European part of Russia

Lichenized fungi as a multicomponent biological system are an interesting but difficult research object. In view of the complex interaction of the myco- and photobiont, the slow growth of natural thalli, their exceptional exactingness to environmental factors, the study of the biological characteristics of lichens is significantly difficult. This article discusses the problem of studying the anatomical and morphological structure of the myco- and photobiont of lichenized fungi using the tissue culture method on the example of rare species (Lobaria pulmonaria) and species with pharmaceutical potential (Usnea dasopoga, Cetraria islandica). The authors proposed a method for cultivating of myco- and photobionts of these species on synthetic nutrient media for the purpose of further research on the characteristics of lichenized fungi. Fragments of thalli, apothecia (for C. islandica), and soredia (for U. dasopoga and L. pulmonaria) were used as donor material. For the introduction of the photobiont under sterile conditions, a homogenate of the thallus region was prepared. Pure cultures of the myco- and photobiont of L. pulmonaria, U. dasopoga, and C. islandica were obtained on three types of hormone-free nutrient media, namely MS nutrient medium, modified MS medium with a reduced nitrogen content, Czapek medium. The verification of the research results was carried out taking into account the microscopy of the obtained cultures of myco- and photobionts. Using microscopy, the dimensional characteristics of the obtained mycobionts were determined. The diameters of the hyphae of L. pulmonaria, U. dasopoga, and C. islandica were 4.3–4.4 µm, 4.6 µm, and 4.1–4.3 µm, respectively, which corresponds to the size of the hyphae in natural samples. The use of the tissue culture method makes it possible to study and analyze the biological characteristics of lichenized fungi as a multicomponent biological system, as well as to contribute to the preservation of rare species and their components in the Red Data Book; while optimizing the method of cultivating myco- and photobiont on synthetic nutrient media, to reduce the anthropogenic load on natural populations of lichens when using them as medicinal raw materials.

\(\textit{In vitro}\) regeneration and acclimatization of \(\textit{Anthurium scherzerianum }\)schott plants

This study investigated the effects of different PGRs on in vitro plant regeneration of Anthurium scherzerianum Schott and acclimatization in the ex vitro stage. Depending on the objective of each experiment, the in vitro shoots were induced from callus cultured on a modified Murashige and Skoog (MS) medium containing cytokinins and auxins. Results showed that 90% of leaf explants and 48% of petiole explants produced callus. A half-strength MS medium supplemented with 1.0 mg/l Kinetin (KIN) gave the best result for shoot regeneration and 1.5 mg/l α-naphthalene acetic acid (NAA) is suitable for rooting of A. scherzerianum. The culture vessel having a hole on the cap gave the best result on the growth of A. scherzerianum plantlets. After 90 days transferred to the greenhouse, in vitro plantlets which were cultivated on a mixed substrate of coconut fiber powder: rice husk ash: compost (6:3:1, respectively) had a better growth (shoot length of 8.88 cm, root length of 3.44 cm) than those on another mixed substrate of coconut fiber powder: compost (9:1, respectively).

Precocious in vitro flowering in threatened ornamental orchid, Dendrobium ovatum – Decoding the causal factors

Dendrobium has been the focus of floral industries for decades. Wild Dendrobiums serve as germplasm for creating hybrids in the cut flower industry and are priced high. But, fewer reports exist on the wild tropical species of Dendrobium. The present study reports in vitro flowering in Dendrobium ovatum - an epiphytic orchid species endemic to the oriental tropics. The seedlings are raised from the protocorms and protocorm-like bodies. This research also highlights the traits displayed by the in vitro flowers of D. ovatum. An optimized modified MS medium was developed to enhance the flowering in the orchid species. The pre-treatment methods, notably dark incubation for 12 h and prechilling at 15 °C for 2 h, demonstrated the synthesis of pharmacologically relevant bioactives viz., ‘Moscatilin’ and ‘Resveratrol’. The tissue culture system devised can decode the enigmatic reproductive biology and its underlying molecular regulation of D. ovatum. This strategy could be emulated in other horticultural, medicinal and food crops for producing industrially relevant phytochemicals.

Callus induced from Stevia rebaudiana Bertoni. leaf used as a substitute of artificial sweetener

Stevia rebaudiana Bertoni is an anti-diabetic plant for which a protocol for rapid plant multiplication from nodal segments and through callus induction from leaf explants was developed with a short incubation period. Maximum callus induction (~100 %) was obtained on modified MS medium incorporated with BAP: NAA (1:3 mg/L) within two to three weeks of incubation under controlled growth room environmental conditions. In the presence of specific concentration/combination of plant growth regulators, the calli obtained from leaf explants were of different colours viz. soft/creamy or off-white, shiny/green, hard/brown and thick/black having varying total sugar content. The present findings deal with induction of callusing in Stevia leaves to achieve rapid callus multiplication. We have also standardized the protocol for plantlet regeneration from Stevia nodal segments where large sized leaves were obtained after 2 weeks of inoculation and the sterile leaves of these regenerated plantlets were used to induce callus. The developed protocol can be utilized for mass production of callus material on a large scale, independent of seasons i.e. external environmental conditions and its dry and purified powdered formulation can be used as a substitute for sugar on a commercial scale.

Efficient regeneration of mature castanopsis hystrix from in vitro stem explants.

Castanopsis hystrix is one of the main timber trees grown in China. However, severe shortage of natural seeds and the difficulty of explant regeneration has limited seedling supply. As such, there is a need for research on asexual multiplication of C. hystrix. This study established a rapid propagation technology system for C. hystrix genotypes, including explant treatment, proliferation, and rooting. HZ (a modified MS medium) supplemented with 4.4 μM BA and 0.5 μM IBA was found to be the optimal medium for shoot sprouting. The maximum proliferation coefficient and the number of effective shoots was obtained on HZ medium supplemented with 2.6 μM BA and 1.0 μM IBA, were 3.00 and 5.63, respectively. A rooting rate of 83.33% was achieved using half-strength HZ medium supplemented with 3.2 μM NAA. Adding vitamin C (80 mg⋅l–1) for 7 days in a dark environment reduced the browning rate, while increasing the proliferation rate. Additionally, through cytological observation, we established how and where adventitious roots occur. The survival rate of transplanted plantlets was > 90%. This is the first report of an in vitro regeneration technique that uses stem segments of mature C. hystrix as explants.

Effects of culture methods and plant growth regulators on in vitro propagation of Brassavola nodosa (L.) Lindl. hybrid

Brassavola nodosa (L.) Lindl. is an epiphytic orchid with great potential for the ornamental plant industry. The lack of information on propagation and production techniques limits the development of large-scale commercial production. Furthermore, this species is experiencing a reduction in population due to habitat destruction, the impact of climate change, and over-collection from native habitats. This study aimed at developing an efficient protocol for micropropagation of B. nodosa, which could be valuable towards the large-scale commercial production as well as for conservation of this species. Six different concentrations of plant growth regulators (BA or IBA, with or without adenine sulfate) were evaluated in modified Murashige and Skoog (MS) medium for shoot multiplication. In addition, two concentrations of either NAA or IBA were evaluated for rooting. Explants were cultured under three different culture media conditions: semi-solid medium, liquid medium (partial immersion), and liquid medium (complete immersion). Results indicate that B. nodosa could be successfully micropropagated in liquid culture with partial immersion. The modified MS medium supplemented with 2.0 mg L−1 BA and 30.0 mg L−1 adenine sulfate resulted in higher multiplication rates. Rooting was obtained using either 0.5 mg L−1 NAA or 1.0 mg L−1 IBA with no significant differences between both rooting treatments. Plantlets achieved 100% ex vitro survival after 30-d acclimatization.

Effect of Silver Nanoparticles on the In Vitro Regeneration, Biochemical, Genetic, and Phenotype Variation in Adventitious Shoots Produced from Leaf Explants in Chrysanthemum.

Novel and unique properties of nanomaterials, which are not apparent in larger-size forms of the same material, encourage the undertaking of studies exploring the multifaced effects of nanomaterials on plants. The results of such studies are not only scientifically relevant but, additionally, can be implemented to plant production and/or breeding. This study aimed to verify the applicability of silver nanoparticles (AgNPs) as a mutagen in chrysanthemum breeding. Chrysanthemum × grandiflorum (Ramat.) Kitam. ‘Lilac Wonder’ and ‘Richmond’ leaf explants were cultured on the modified MS medium supplemented with 0.6 mg·L−1 6-benzylaminopurine (BAP) and 2 mg·L−1 indole-3-acetic acid (IAA) and treated with AgNPs (spherical; 20 nm in diameter size; 0, 50, and 100 mg·L−1). AgNPs strongly suppressed the capability of leaf explants to form adventitious shoots and the efficiency of shoot regeneration. The content of primary and secondary metabolites (chlorophyll a, chlorophyll b, total chlorophylls, carotenoids, anthocyanins, phenolic compounds) and the activity of enzymatic antioxidants (superoxide dismutase and guaiacol peroxide) in leaf explants varied depending on the AgNPs treatment and age of culture. Phenotype variations of ex vitro cultivated chrysanthemums, covering the color and pigment content in the inflorescence, were detected in one 50 mg·L−1 AgNPs-derived and five 100 mg·L−1 AgNPs-derived ‘Lilac Wonder’ plants and were manifested as the color change from pink to burgundy-gold. However, no changes in inflorescence color/shape were found among AgNPs-treated ‘Richmond’ chrysanthemums. On the other hand, the stem height, number of leaves, and chlorophyll content in leaves varied depending on the AgNPs treatment and the cultivar analyzed. A significant effect of AgNPs on the genetic variation occurrence was found. A nearly two-fold higher share of polymorphic products, in both cultivars studied, was generated by RAPD markers than by SCoTs. To conclude, protocols using leaf explant treatment with AgNPs can be used as a novel breeding technique in chrysanthemum. However, the individual cultivars may differ in biochemical response, the efficiency of in vitro regeneration, genetic variation, and frequency of induced mutations in flowering plants.

Elaboration of micropropagation system of medicinal plant yacon (Smallanthus sonchifolius (Poepp.) H. Rob.)

Yacon becomes an important plant with strong medical and dietary potential. Therefore, studies on the optimization of yacon micropropagation were undertaken. Reproduction of yacon is conducted by traditional methods with green seedlings or long offsets taken from the underground and aboveground rootstock. The influence of different disinfection methods, various initial explants and growth regulators, their concentrations, and combinations on the efficiency of micropropagation have been analyzed. The most effective disinfection was the use of 70% ethanol and 0.5% Tween 20. The optimal initial explants were apical and lateral buds cultured on Murashige and Skoog (MS) medium with 0.2 mg L–1 kinetin and 1 mg L–1 indole-3-acetic acid. The most intensive shoot regeneration occurred on MS medium with 1 mg L–1 thidiazuron and 1-naphthylacetic acid. Histological observations demonstrated the strong effect of growth substances on shoot organogenesis from callus tissue. Shoots obtained from in vitro cultures rooted easily on modified MS media with ½ or ¼ concentration of macroelements, 20 g L–1 sucrose and 3 mg L–1 1-naphthylacetic acid. All plants adapted to ex vitro conditions.

New insights into the role of chrysanthemum calcineurin B\u2013like interacting protein kinase CmCIPK23 in nitrate signaling in Arabidopsis roots

Nitrate is an important source of nitrogen and also acts as a signaling molecule to trigger numerous physiological, growth, and developmental processes throughout the life of the plant. Many nitrate transporters, transcription factors, and protein kinases participate in the regulation of nitrate signaling. Here, we identified a gene encoding the chrysanthemum calcineurin B-like interacting protein kinase CmCIPK23, which participates in nitrate signaling pathways. In Arabidopsis, overexpression of CmCIPK23 significantly decreased lateral root number and length and primary root length compared to the WT when grown on modified Murashige and Skoog medium with KNO3 as the sole nitrogen source (modified MS). The expression of nitrate-responsive genes differed significantly between CmCIPK23-overexpressing Arabidopsis (CmCIPK23-OE) and the WT after nitrate treatment. Nitrate content was significantly lower in CmCIPK23-OE roots, which may have resulted from reduced nitrate uptake at high external nitrate concentrations (≥ 1 mM). Nitrate reductase activity and the expression of nitrate reductase and glutamine synthase genes were lower in CmCIPK23-OE roots. We also found that CmCIPK23 interacted with the transcription factor CmTGA1, whose Arabidopsis homolog regulates the nitrate response. We inferred that CmCIPK23 overexpression influences root development on modified MS medium, as well as root nitrate uptake and assimilation at high external nitrate supply. These findings offer new perspectives on the mechanisms by which the chrysanthemum CBL interacting protein kinase CmCIPK23 influences nitrate signaling.

Response of wheat genotypes to excess boron estimated by in vitro culture

The objective of this study was to evaluate boron tolerance of wheat genotypes using mature embryo culture. The analysis involved 79 recombinant inbred lines of the International Triticeae Mapping Initiative (ITMI) population and three Serbian varieties with known boron (B) tolerance (Pobeda - sensitive S, Balerina-medium tolerant MT, and Nevesinjka - tolerant T). The evaluation was performed on a modified MS medium to which 15 mM of boric acid was added. The control medium contained no excess B. Callus fresh weight (CFW) and reduction of fresh callus weight (RFCW) were determined after one month of cultivation. ANOVA has shown highly significant effect of genotype, the media, and their interaction to callus tissue growth and also significant genotypic effect on RFCW. Majority of genotypes (39) had sensitive reaction to excess boron, twenty-three were medium tolerant, while four of them were tolerant. The obtained results can be potentially used for mapping QTLs associated with tolerance to excess B in wheat breeding program.

Effect of Nitrogen on in Vitro Propagation of Endangered Medicinal Plant: Swertia Chirayita Roxb. Ex Flaming

The effect of nitrogen was investigated on the organogenesis of Swertia chirayita (Gentianaceae) to overcome the challenges related to its cultivation. The best callogenic response was observed on root explants inoculated onto MS medium supplemented with BAP (2.0 mg/l) along with 2,4-D (0.5 mg/l) after 35 days of culture. Subsequent transfer of callus for multiplication on the same media composition under complete darkness presents the best results in terms of callus multiplication. Callogenic cultures were subculture onto modified MS medium supplemented with inorganic nitrogen sources, i.e., NH4NO3 (14-56N/l), KNO3 (100-400N/l) with BAP (3.0 mg/l) were observed. Organogenic response (52%) was observed after 8-12 weeks of culturing. The maximum number of the shoot was recorded on MS medium with NH4NO3 (28 N/l), KNO3 (300N/l) with BAP (3.0 mg/l). Moreover, 90% of them were able to regrow when sub-cultured on the same media. Sixteen weeks old multiple shoots were subcultured on MS medium supplemented with different auxins. IAA was proved to be the best hormone rooting purpose. However, the best rooting response regarding the number of roots and an average length of roots was obtained at IAA (1.0 mg/l). Survival of 90% was achieved when rooted plantlets were successfully established in substrate containing sand, vermicompost, and garden soil in equal proportion for hardening and acclimatized.

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)

Production of Virus-Free Garlic Plants through Somatic Embryogenesis

The present study was conducted to establish a protocol for the regeneration of virus-free garlic plants through somatic embryogenesis of two Croatian garlic ecotypes. Basal parts of cloves from mother plants were cultured on a full Murashige and Skoog (MS) or modified MS medium (¼ of KNO3 and NH4NO3 and 2xMgSO4) containing 0.1 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) or 1 mg L−1 2,4-D + 0.5 mg L−1 kinetin (Kin) and representing four different treatments. Plants were regenerated in MS medium containing 0.1 mg L−1 2,4-D and rooted in a medium containing 0.05 mg L−1 1-naphthaleneacetic acid (NAA) + 0.005 mg L−1 6-(γ,γ-dimethylallylamino)purine (2iP). The presence of viruses (i.e., sanitary status) of the mother plants and regenerants was checked by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). The mother plants were infected with onion yellow dwarf virus (OYDV) and leek yellow stripe virus (LYSV). In addition, the presence of garlic common latent virus (GCLV) was confirmed in four mother plants. Embryogenic callus developed in all four treatments with success ranging from 55% to 81% depending on treatment and ecotype. Plant conversion was significantly higher in somatic embryos developed in media containing 0.1 mg L−1 2,4-D than those developed in media containing 1 mg L−1 2,4-D + 0.5 mg L−1 Kin. Virus elimination success ranged from 13.3% up to 62.5% depending on garlic ecotype and treatment. The overall rate of virus elimination by somatic embryogenesis for both treatments and ecotypes were 20.7%, 22.9%, and 30.5% for OYDV, GCLV, and LYSV, respectively. Based on these results, somatic embryogenesis has been shown to be equally or more successful in eliminating garlic viruses compared to other in vitro methods.

Tracheary elements from calli of Japanese horse chestnut (Aesculus turbinata) form perforation-like structures.

Calli derived from young leaves of Aesculus turbinata contained tracheary elements with large pores that resembled perforations of vessel elements. The differentiation of tracheary elements in vitro provides a useful system for detailed analysis of xylem cell differentiation. To examine the mechanism of formation of cell wall structures, new differentiation systems are required that allows us to induce highly organized structures, such as perforations. In this study, we developed such a system in which we were able to induce formation of tracheary elements with perforations, using calli of a hardwood, Aesculus turbinata. Young leaves of A. turbinata were placed on modified MS medium that contained 5μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5μM benzyladenine (BA). Tracheary elements were induced in calli derived from young leaves of A. turbinata. Some tracheary elements formed broad areas of secondary wall with typical features of secondary xylem. Other tracheary elements formed spiral thickenings, which are typical features of vessel elements in secondary xylem of A. turbinata. Approximately 10% of tracheary elements formed large pores that resembled perforations of vessel elements and various types of the perforation plate were observed. Addition of NAA and brassinolide to the induction medium enhanced the differentiation of tracheary elements in calli of A. turbinata. Newly induced tracheary elements also formed typical features of secondary xylem such as perforations of the vessel elements. Our model system might be useful in efforts to understand the mechanisms of formation of highly organized structures in tracheary elements in secondary xylem.

Induksi Kalus Morfogenik dan Regenerasi Tanaman Tebu (Saccharum officinarum L.) secara In Vitro

Propagasi in vitro merupakan cara memperoleh tanaman baru yang sehat secara cepat dan massal, Percobaan in vitro dilaksanakan untuk menginduksi kalus morfogenik dan regenerasi dari varietas tebu domestik. Bagian terdalam gulungan daun tebu digunakan sebagai eksplan dan diinkubasi pada medium MS yang dimodifikasi dan dilengkapi dengan 2,4-D pada konsentrasi 1 mg/l, 2.5 mg/l, dan 5 mg/l untuk menginduksi pembentukan kalus. Kalus yang terbentuk di medium induksi selanjutnya dipindahkan ke medium MS yang diperkaya dengan zat pengatur tumbuh untuk inisiasi dan perbanyakan tunas. Persentase pembentukan kalus tertinggi dicapai pada penggunaan medium yang mengandung 2.5 mg/l 2,4-D dan maksimal proliferasi kalus morfogenik diperoleh setelah subkultur ketiga. Dua tipe kalus yang terbentuk pada medium induksi yaitu berstruktur kering, remah dan struktur halus kompak. Jenis kalus yang morfogenik tersebut berwarna putih dan putih kekuningan serta mudah terpisah-pisah. Hasil proliferasi tunas dari kalus tertinggi diperoleh pada medium MS dengan kombinasi hormon tumbuh 2 mg/l Kinetin + 1 mg/l IAA dan tidak ada pertumbuhan tunas pada medium yang ditambahkan zat pengatur tumbuh tunggal, yaitu Kinetin. Dengan demikian, penelitian ini menyimpulkan bahwa hormon tumbuh jenis sitokinin yang dikombinasi dengan auksin sangat dibutuhkan untuk regenerasi kalus tebu secara in vitro.

Optimizing plant regeneration and genetic transformation of Paulownia elongata

The effects of plant tissue culture media with varying levels of growth regulators and carbon sources was tested on four explant types for organogenesis and plant regeneration in Paulownia elongata. Among the various treatments applied, the best response to adventitious shoot proliferation was observed when entire leaf or half leaf with attached petiole explants were cultured on either MS or B5 medium along with 25 μM thidiazuron, 10 μM indole acetic acid and 30 g L−1 maltose as a carbon source. Proliferated shoots were rooted within five days on a modified MS medium containing 5 μM indole butyric acid and plants were successfully acclimated and hardened for greenhouse transfer. A green fluorescent protein (gfp) and neomycin phosphotransferase II (npt II) gene was used to optimize genetic transformation protocols. Agrobacterium tumefaciens mediated transformation of half leaf with petiole explants produced transgenic plants using the media optimized for shoot proliferation and rooting. The insertion and expression of the GFP transgene was detected using PCR and RT-PCR.

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.

Effect of media concentration and growth hormones on shoot regeneration and in vitro rooting of sugarcane varieties (Saccharum spp. )

The protocol for plantlet induction and regeneration through in vitro culture was standardized for the sugarcane variety Co 06022 with Co 86032. Genotype specific media combinations for establishment and rooting efficiency was identified and standardized. Multiple shoot regeneration was observed at different frequencies, using different concentration and growth regulator combination. In vitro propagation of Saccharum offers opportunities for increasing plant material for cultivation. Apical meristems were cultured on modified MS medium containing different concentrations of auxins. In Co 06022, the most effective concentration was combination of ½ MS medium supplemented with IBA (2.0 mg/l) which induced an average of 3.00±0.14 number of shoots per microshoot with 10.77±0.42 cm shoot length. Among the auxins used, NAA showed better response than IBA for profuse rooting in Co 06022. The number of roots formed was maximum (14.80±0.09) in MS with NAA (2mg/l) which had taken 10 days for root initiation with the root length of 6.45±0.07 cm. However, in Co 86032, the best performance was observed on ½ MS medium supplemented with IBA (2.0 mg/l) with more number of shoots (2.00 ± 0.19) and average shoot length of 12.30± 0.44 cm. Best rooting was observed in ½ MS medium supplemented with NAA (2.0 mg/l) and maximum number of roots were 11.60±0.12 which has taken 8 days for initiation of root primordia with average root length of 1.16±0.12 cm. Thus, it can be deduced that this protocol can be used successfully for in vitro rooting and acclimatization of these genotypes.