μM NAA Research Articles

Shoot organogenesis and somatic embryogenesis from leaf and petiole explants of endangered Euryodendron excelsum

Euryodendron excelsum H.T. Chang is a rare and endangered woody plant endemic to China. It is very important to conserve and propagate this species from extinction. In this study, leaves and petioles from the axillary shoots in vitro were used as explants to culture on the different plant growth regulator (PGR) woody plant medium (WPM) and establish an efficient shoot proliferation and plant regeneration system. WPM supplemented with 1.0 mg/L 2,4-D induced callus dedifferentiated into buds and somatic embryos on various media,including PGR-free WPM. However, only adventitious shoots formed on WPM with 1.0 mg/L of cytokinins such as 6-benzyladenine (BA), kinetin (KIN) or thidiazuron (TDZ). When another cytokinin, zeatin, was used, somatic embryos were induced directly from From cut surface of these explants. Adventitious roots could be induced from both explants on WPM with 1.0 mg/L α-naphthaleneacetic acid (NAA). Somatic embryos cultured in PGR-free WPM or WPM with 0.2 mg/L NAA developed roots. Plantlets derived from somatic embryos were transferred to a peat: sand (1:1, v/v) substrate, and showed survival rates of 64.3% at 30 days and 54.6% at 90 days. Callus clumps with adventitious shoot buds that were transferred to WPM containing 1.0 mg/L BA and 0.2 mg/L NAA generated a mean 3.3 multiple shoots. Callus-derived shoots regenerated and rooted successfully (100%) on agar-free vermiculite-based WPM with 0.5 μM NAA after 30 d. Plantlets transplanted to peat soil: vermiculite (1:1, v/v) displayed the highest survival (96.7%) after three months.

High‑frequency plant regeneration and genetic fidelity assessment of regenerants by molecular and biochemical markers in Woodland Sage (Salvia nemorosa L.)

Salvia nemorosa L. is a valuable medicinal and ornamental plant from Lamiaceae . A high-frequency protocol for in vitro regeneration from S. nemorosa leaf explants was developed based on our results. Various plant growth regulators were evaluated for their effects on direct and indirect organogenesis , callogenesis, shoot multiplication, and rooting of explants at different concentrations. The maximum percentage of direct shoot regeneration (97.5%) and the number of shoots per explant (8.6) were obtained on MS medium supplement with 8 µM TDZ plus 8 µM NAA . The result revealed that media amended by 16 µM 2, 4-D with 8 or 16 µM BA was the best treatment for callus induction. Media containing the combination of 8 µM TDZ plus 2 µM NAA provided the highest rate of shoot induction (84%) and the highest number of shoots (5.4) from calli. Regenerated shoots were multiplied efficiently with an average of 7.4 shoots and 32.4 leaves per explant on media containing 12 µM TDZ. In the present study, TDZ and BA treatments led to the highest proliferation rate; however, adenine sulfate was preferred for proliferation because it did not produce hyperhydric plants. The genetic fidelity of 19 plantlets (one donor plant, nine direct and nine indirect regenerants) was appraised by 10 ISSR markers. The molecular analysis revealed that direct organogenesis produced true-to-type plantlets and could be employed for clonal multiplication of Woodland Sage. Indirect regenerants showed polymorphic bands (12.5–33.33%), confirming the somaclonal variation occurrence during indirect organogenesis. Also, HPLC analysis of regenerants showed that two indirect regenerants accumulated higher rosmarinic acid content than mother plants, while three of them accumulated lower rosmarinic acid content. As part of this study, molecular and biochemical markers have been employed to investigate somaclonal variation in in vitro regeneration of the Salvia genus for the first time, which could be utilized to develop reliable methods for multiplying elite lines and achieving high-yield lines. • A high-frequency protocol for S. nemorosa in vitro regeneration was developed. • TDZ and NAA combination produced plantlets with high genetic and biochemical fidelity. • Adenine Sulphate prevents the Hyperhydricity in regenerated plantlets. • The indirect regeneration affected PAL activity, rosmarinic acid, and genetic content.

Effects of α-Naphthylacetic Acid on Cadmium Stress and Related Factors of Tomato by Regulation of Gene Expression

Cadmium (Cd) is absorbed and accumulated by crops, and it adversely affects plant growth and development. To explore the effect of exogenous auxin on Cd stress, we applied different concentrations of α-naphthaleneacetic acid (NAA) and the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) to tomato plants exposed to Cd stress in a hydroponic system. NAA and TIBA at different concentrations were used under Cd stress. Plant growth, root morphology, and auxin distribution were observed. Lipid peroxidation and antioxidant enzyme activities in leaves, cadmiumcontent, and migration coefficient of plants were determined. Transcriptome sequencing and qRT-PCR were used to analyze the differentially expressed genes. Results showed that auxin was concentrated in the leaf veins, stem base, and roots in P5::GUS “Chico III” transgenic tomato, indicating NAA polar transport to the roots and promotion of root growth under Cd stress. Cd was absorbed by the roots and transported to the shoots. It then inhibited plant growth and promoted antioxidant enzyme activities, O2− production, H2O2 accumulation, and membrane lipid peroxidation. Treatment with 0.5 μM NAA improved antioxidant enzyme activities, reduced reactive oxygen, maintained membrane permeability, and decreased malondialdehyde and proline contents. Transcriptome analysis revealed that NAA activated a large number of genes in the roots: 1998 genes were differentially expressed in response to Cd or NAA treatment, and 1736 genes were specifically expressed in response to NAA treatment under Cd stress. Among the differentially expressed genes, tomato metallocarboxypeptidase inhibitor TCMP-2 (2A11) and Solanum lycopersicum heavy metal-associated isoprenylated plant protein (HIPP) 7-like (LOC101264884), which are closely related to plant response to heavy metal stress, may be the key sites of NAA. In conclusion, the NAA-mediated response to Cd stress was closely associated with “defense response” genes in shoots and “oxidoreductase activity, oxidizing metal ions” and “response to auxin” genes in roots.

Micropropagation, encapsulation, physiological, and genetic homogeneity assessment in Casuarina equisetifolia.

Casuarina equisetifolia is an important tree of the forest, cultivated in tropical and subtropical regions, providing fuelwood, land reclamation, dune stabilization, paper production, and nitrogen fixation. We have developed a systematic in vitro propagation protocol in C. equisetifolia using nodal segments (NS). Murashige and Skoog (MS) medium augmented with BA (5.0 μM) and NAA (0.5 μM) gave rise to a maximum of 32.00 ± 0.31 shoots per explant (S/E) with shoot length (SL) of 3.94 ± 0.02 cm, and a maximum of 70% regeneration potential (RP) was recorded after 8 weeks of post inoculation. For root induction, in vitro derived shoots were transferred to the nutrient medium consisting of a half-strength (½) MS medium augmented with 2.5 μM NAA, which produced a maximum of 12.68 ± 0.33 roots/shoot (R/S) with 3.04 ± 0.50 cm root length (RL) in 60% of culture after 6 weeks. Micropropagated plants with healthy shoots and roots were successfully acclimatized in vermicompost + garden soil + sand (1:2:1) and a maximum survival percentage of 95.1% was recorded. NS was taken from a 6-weeks-old in vitro derived plant of C. equisetifolia for synthetic seed production, and it was reported that CaCl2 · 2H2O (100 mM) + Na2-alginate (4%) resulted in clear and uniform beads. Furthermore, the maximum conversion of synthetic seeds into plantlets occurred over a period of 4 weeks of storage at 4°C. Scanning Electron Microscopy (SEM) revealed the formation of direct shoot buds without any intermediate callus formation. In addition, the chlorophyll and carotenoid contents of the direct regenerated and mother plant were compared. Similarly, RAPD and ISSR primers were used for genetic homogeneity assessment of the direct regenerated plants, where a total of 18 and 19, respectively, clear and reproducible bands with 100% monomorphism were recorded. The developed micropropagation protocol can certainly be used for large-scale multiplication and germplasm preservation of C. equisetifolia. It will also help in meeting the growing demands of C. equisetifolia in the forest industry.

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.

Establishment of an Efficient In Vitro Propagation Method for a Sustainable Supply of Plectranthus amboinicus (Lour.) and Genetic Homogeneity Using Flow Cytometry and SPAR Markers

Plectranthus amboinicus (Lour.) Spreng is a medicinally important aromatic perennial herb used for the treatment of skin diseases, constipation, asthma, flu, fever, cough, and headache as well as a flavoring ingredient in traditional drinks, food, and meat stuffing. In this study, a high-performance in vitro propagation system of P. amboinicus through direct shoot organogenesis was developed using axillary node explants cultured on MS (Murashige and Skoog) medium augmented with 0.5, 2.5, 5.0, 7.5, and 10.0 µM of 6-benzyladenine (BA) or kinetin (Kin), alone or with 0.1, 0.5, 2.5, and 5.0 µM of indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA). To optimize the regeneration potential of node explants, the effects of basal media strength and pH were also investigated. After 8 weeks of culture, explants cultured in full strength MS basal medium (pH 5.7) with 5.0 µM BA and 2.5 µM NAA exhibited the highest percentage (97.1%) of regeneration and the maximum number (19.3) of shoots per explant. Individual elongated shoots were rooted on half strength MS basal medium containing 0.25 µM indole 3-butyric acid (IBA) after 4 weeks of culture, producing 5.3 roots/shootlets with a root induction frequency of 93.7%. First time genetic stability of in vitro raised P. amboinicus plants was determined using SPAR markers, such as DAMD and ISSR, as well as flow cytometric tests, assuring the availability of authenticated raw materials for commercial production of the plant and its bioactive components.

Induction of somatic embryogenesis in Tecomella undulata (Sm.) Seem using a pistillate explant

Desert teak (Tecomella undulata (Sm.) Seem) a multipurpose ornamental tree native to the arid and semi-arid tropics has entered the endangered plant category mainly as a result of the species' ineffective seed reproduction system. The tree usually reproduces through a few root suckers in old stands. Conventional methods of plants multiplication could not offer a viable practice for its mass multiplication. Low adventitious rooting of the cuttings has been the principal cause of failure in its vegetative propagation. Hence, the present research was planned and conducted to evaluate the feasibility of somatic embryogenesis in this species, the pathway that bypasses the need for rooting stage by developing bipolar embryos. Ovary explant was cultured in modified Murashigue & Skoog (MS) medium supplemented with different auxins and cytokinins. The results showed α-naphthalene acetic acid (NAA) was superior in inducing embryogenic callus. NAA ranging 5.4-21.5 μM could induce the highest embryogenic calli which exhibited developing pro-embryogenic masses (PEM) and globular somatic embryos. The calli which were induced by the use of 2,4-dichlorophenoxyacetic acid (2,4-D) were poor in quality and showed no morphogenesis potency. Individual application of Thidiazuron (TDZ) and N6-benzyladenine (BA) induced good callogenesis at low concentrations but the calli were non-embryogenic with both. The proliferation of embryogenic calli was the best in a hormone-free medium. However, the media containing 40.5 and 54 μM NAA alone could induce somatic embryos along with callus proliferation. Low BA-contained medium (0.9-4.44 μM) led to recurrent somatic embryogenesis. Neither BA and GA3, nor the elevating sucrose concentration could cause further development and maturation of the somatic embryos induced during previous stages (callogenesis and callus proliferation). More research is required to optimize the maturation stage. The findings of the present study can be useful for future studies in the micropropagation of this recalcitrant specieslationships in Indian mustard under heat stress and the differential remobilization efficiencies in the advanced breeding lines.

An Improved Procedure for Agrobacterium-Mediated Transformation of 'Carrizo' Citrange.

Although several protocols for genetic transformation of citrus have been published, it is highly desirable to further improve its efficiency. Here we report treatments of Agrobacterium cells and citrus explants prior to and during co-cultivation process to enhance transformation efficiency using a commercially used rootstock ‘Carrizo’ citrange [Citrus sinensis (L.) Osb. × Poncirius trifoliata (L.) Raf.] as a model plant. We found explants from light-grown seedlings exhibited higher transformation efficiency than those from etiolated seedlings. We pre-cultured Agrobacterium cells in a 1/10 MS, 0.5 g/L 2-(N-morpholino) ethanesulfonic acid (MES) and 100 µM acetosyringone liquid medium for 6 h at 25 °C before used to infect citrus explants. We incubated epicotyl segments in an MS liquid medium containing 13.2 µM 6-BA, 4.5 µM 2,4-D, 0.5 µM NAA for 3 h at 25 °C prior to Agrobacterium infection. In the co-cultivation medium, we added 30 µM paclobutrazol and 10 µM lipoic acid. Each of these treatments significantly increased the efficiencies of transformation up to 30.4% (treating Agrobacterium with acetosyringone), 31.8% (treating explants with cytokinin and auxin), 34.9% (paclobutrazol) and 38.6% (lipoic acid), respectively. When the three treatments were combined, we observed that the transformation efficiency was enhanced from 11.5% to 52.3%. The improvement of genetic transformation efficiency mediated by these three simple treatments may facilitate more efficient applications of transgenic and gene editing technologies for functional characterization of citrus genes and for genetic improvement of citrus cultivars.

ENHANCED PRODUCTION OF VASICINE, AN ALKALOID IN CALLUS AND SUSPENSION CULTURES OF Adhatoda zeylanica

Adhatoda zeylanica is an important medicinal plant and being used in the rural areas of India for several ailments. It belongs to the family Acanthaceae and is known as Vasaka in Sanskrit, Malabar nut in English. The leaves contain a pyrroloquinazoline alkaloid called Vasicine. It is a powerful expectorant, antiseptic, antiperiodic and antihelminthic. Besides seasonal variations, majority of the plant-based alkaloids can not be chemically synthesized. Tissue culture protocols for a number of medicinal plants were established, and this enabled the analysis of callus/cell suspension extracts for the presence of different classes of secondary products.. Therefore, research attempts for the production of secondary metabolites of pharmaceutical importance through tissue culture approach was taken up to enhance the production of vasicine. Callus cultures were established from leaves and petioles of A.zeylanica on Murashige and Skoog and B5 medium supplemented with auxins (2,4-D and NAA) and cytokinins (6-BA and kinetin).The synthesis of vasicine was more in leaf-derived callus grown on MS medium with 10.7µM NAA and 4.4 µM 6-BA along with 0.5ml/l of Ornithine(4.7mg/gDW). Suspension cultures were treated with different biotic and abiotic elicitors to increase the content of vasicine. Aspergillus niger at a concentration of 1.5ml/l gave maximum yield of 4.72mg/gDW of vasicine after an incubation of 48hrs. Chitosan yielded 5.54mg/gDW of vasicine at a concentration of 150mg/l for 48 hrs incubation.Synthesis of vasicine was detected and confirmed by thin layer chromatography,1H NMR, 13C NMR, electron ionization mass spectra and high pressure liquid chromatography analyses.

Acceleration of micropropagation procedure of Bambusa nutans: A commercially important bamboo species

The present communication described an accelerated, efficient and reproducible in vitro propagation procedure for large-scale multiplication of Bambusa nutans Roxb. The nodal segments (12–18 mm) collected from secondary branches of superior clump in the month of April reported enhanced multiple shoot formation (7-8) when implanted on Murashige & Skoog semi solid medium supplemented with 10 µM BA (6-Benzylaminopurine). These axillary shoot cultures pass through ten subculture cycle on liquid medium of 10 µM BA. The cluster of two axillary shoots transferred to liquid MS medium containing 10 µM BA+ 7.0 µM NAA (a-Naphthaleneacetic acid) produced a maximum of 14.5 multiple shoots after 20 days of inoculation. Rooting of 100 % of shoots was achieved in excised propagules when transferred to ½ MS medium supplemented with 30 µM NAA. A two-step hardening procedure of in vitro raised plants ensured 100 % survival. Macroproliferation was carried out in hardened plants after 4 months registered threefold number of plants in polythene bags. The field transferred plants were reported with 100 % survival with prolific new shoot formation and growth.

Efficient micropropagation of Thunbergia coccinea Wall. and genetic homogeneity assessment through RAPD and ISSR markers

Thunbergia coccinea Wall. ex D. Don being a rare, ornamental and medicinal plant of India, is needed to propagate for conserving the germplasm and analyzing its phytochemical compounds in the future. A reliable protocol for direct in vitro propagation using nodal shoot meristem of T. coccinea as explant was standardized. The highest number of shoots per explant (22.17 ± 0.54) with maximum shoot length (2.36 ± 0.28) in cm was obtained in Murashige and Skoog (MS) medium supplemented with 9.70 µM of 6-furfurylaminopurine (Kinetin) and 0.053 µM of α-naphthaleneacetic acid (NAA) combination, among all the different plant growth regulators (PGR’s) and concentrations tested. The aforesaid PGR’s combination was optimum for axillary shoot bud induction and multiplication in T. coccinea. The best rooting was observed on the half-strength MS medium fortified with 2.68 µM NAA with the highest number of roots per shoot (3.75 ± 0.12) and maximum length (5.22 ± 0.32) in cm. All the in vitro raised plantlets were acclimatized in sterile sand and soil mixture (1:1) with a survival rate of 70% on earthen pots under greenhouse conditions. PCR-based RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeat) molecular markers were employed to determine the genetic homogeneity amongst the plantlets. Twelve (12) RAPD and nine (9) ISSR primers developed a total of 104 and 91 scorable bands, respectively. The band profiles of micropropagated plantlets were monomorphic to the mother, donor in vivo plant, and similarity values varied from 0.9542–1.000. The dendrogram generated through UPGMA (unweighted pair group method with arithmetic mean) showed 99% similarities amongst all tested plants confirming the genetic uniformity of in vitro raised plants.

In vitro Regeneration of Japanese Burdock (Arctium lappa L.) from Cotyledon and Leaf Derived Explants

Considering the vegetable and medicinal values, a micropropagation protocol has been established for Japanese Burdock (Arctium lappa L.) by culturing the explants of cotyledon and leaf obtained from in vitro grown seedlings. Direct shoot regeneration was achieved from cotyledon and leaf explants on MS fortified with 4.0 μM BAP and 2.0 μM IBA or NAA after 5 weeks of culture. In addition, both the explants also formed callus from their cut margins within 6 weeks of cultivation on medium complemented with 6.0 μM BAP and 4.0 μM IBA or NAA. Adventitious shoots were also redeveloped through indirect organogenesis from the cotyledon and leaf-derived callus within 10 weeks of culture on MS containing 4.0 μM BAP and 2.0 μM IBA or NAA. The highest rate of shoot reproduction was attained at the third subculture, and more than 12.6 shoots were formed per callus clump. Within 4 weeks of transfer to the rooting medium on MS containing 6.0 μM IBA, the cultured micro-shoots produced highest 5.3 roots per cultured shoot. Rooted plantlets were successfully established on a soil-composed-sand mixture under natural condition with 93.3% survival rate Plant Tissue Cult. & Biotech. 31(2): 123-134, 2021 (December)

Pengaruh BAP dan NAA terhadap Waktu Pertumbuhan Tanaman Eceng Gondok (Eichhornia crassipes) secara In Vitro

Eichhornia crassipes (a carnivorous plant) is an endemic carnivorous plant of Mount Tangkuban Perahu, which is listed the convention on international trade of endangered species (CITES). One potential posses Eichhornia crassipes the Biological control of insects and plants medicine. The objectives of this study were to know concentration BAP and NAA optimal and know the time appear the emergence of shoots, roots and leaves. This research has been carried out experimentally. That stage of growth in bring up shoots, roots and leaves use a Completely Randomised Design on a two factors factorial pattern was used. The first factor was BAP concentrations with 4 levels 0 µM, 5 µM, 10 µM, and 15 µM. The second factor was NAA concentration with 4 levels 0 µM, 0,5 µM, 1 µM, and 1,5 µM. The research results showed germination of seeds in plants Eichhornia crassipes using media vacin and went (VW) with the addition of substance of a substance controller of growing BAP 4 µM produce quite a lot germination and treatment 10 µM BAP and 0,5 µM NAA is the fastest in time to occur , appear leaves and appear roots.

In Vitro Regeneration of Muscari racemosum Mill. Using Twin Bulb Scales, Primary Bulbs, and Leaf Bases

Turkey is an important center of diversity for many plants species including bulbs, rhizomes, tubers, and other plants of high agricultural and horticultural importance. These species have a special importance as ornamental plants. However, due to urbanization and related factors, many of them are under threat. One of these species is the endemic Muscari racemosum Mill. The current study aimed to develop an efficient in vitro commercial bulblet propagation procedure using different explants. Twin-scale bulb explants were regenerated on MS medium having several doses of Kinetin+NAA (1-Naphthaleneacetic acid). The best regeneration was exhibited on 4.65 μM Kinetin+5.37 μM NAA at the end of 10 weeks with induction of 4.08 bulblets/explant with a mean diameter of 0.31 cm. The primary bulblets were cultured on MS medium having 18.60 μM Kinetin+5.37 μM NAA. About a 2.5-fold increase in the diameter of the bulbs (0.76 cm) was exhibited on the regenerated bulblets. The bulblets were regenerated on leaf bases taken from MS medium having several doses of BAP (6-Benzylaminopurine) + NAA. The regenerated bulbs were rooted on MS medium having 4.90 μM IBA (Indole-3-butyric acid) followed by their transference to a greenhouse for acclimatization. This study provided important information on commercial clonal propagation of M. racemosum and the importance of explants and growth regulators in plant regeneration.

Promotion of flower opening in cut rose cultivars by 1-naphthaleneacetic acid treatment

ABSTRACT Improving the quality and rate of opening of cut flowers is important to meet consumer demand. Thus, it is important to develop methods to control the rate of flower opening and senescence in ornamental plants. In this study, we investigated the effects of 1-naphthaleneacetic acid (NAA) in flower opening in rose (Rosa sp.) cultivars Princess Meg, Red Star and Madrid. Cut roses were maintained under different concentrations of NAA. Shoot bases were immersed in water solution containing 0, 100, and 1,000 μM NAA, in addition to 2% w/v sucrose with 0.02% w/v 8-hydroxyquinoline monohydrate. Subsequently, their vase life, flower opening, flower diameter and petal weight were measured. Flower opening in all three cultivars was clearly promoted by the 1,000 μM NAA treatment, resulting in higher petal fresh weight and flower diameter at 2 days following treatment. 100 μM NAA treatment also promoted flower opening and petal wilting in three cultivars, although the decrease in relative fresh weight of cut rose became slower and vase-life became longer than 1,000 μM NAA treatment in “Madrid”. This indicates that NAA promotes flower opening and petal growth in three cut rose cultivars. However, NAA treatment also promoted petal wilting, resulting in shorter vase-life. Although rose cultivars differed in their sensitivity to the NAA treatment, we conclude that NAA shows high potential as a chemical agent for controlling flower opening in cut rose cultivars.

Dynamics of Polyphenol Biosynthesis by Calli Cultures, Suspension Cultures and Wild Specimens of the Medicinal Plant Ligaria cuneifolia (Ruiz & Pav.) Tiegh. (Loranthaceae). Analysis of Their Biological Activity.

Ligaria cuneifolia (R. et P.) Tiegh. (Loranthaceae) is a South American hemiparasitic species with antioxidant, antitumoral, antimicrobial, and antilipidemic activities attributed to its polyphenolic content. We studied the polyphenolic pattern of L. cuneifolia during different phenological stages: flowering, fruiting, and post-fruiting. The highest total phenolic content was found in stems at post-fruiting (214 ± 12.1 mg gallic acid eq·g−1 DW) and fruiting (209 ± 13.7 mg gallic acid eq·g−1 DW), followed by post-fruiting leaves (207 ± 17.5 mg gallic acid eq·g−1 DW). Flavonoids accumulated at higher levels in leaves and hydroxycinnamic acids in leaves at flowering and post-fruiting. The polyphenolic pattern was similar between organs from wild plants and in vitro cultures, although at a significantly lower level in the latter ones. The performance of calli growing under a 16 h photoperiod in a modified White medium with 1-naphthalene acetic acid (2.50 μM) and Kinetin (9.20 μM) was better than in the dark. When calli grew in media only with auxins (IAA, NAA, and 2,4-D, all at 2.50 µM concentration), its growth and polyphenolic content improved. Cell suspensions with 2.50 µM NAA and 9.20 µM KIN grew slowly and produced very small amounts of polyphenols. As for the antioxidant activity, it was detected in all samples (approximately 1000 µmol trolox eq·g−1 DW) except fruits, where a lower value was found (328 µmol trolox eq·g−1 DW). In vitro cultures have the lowest antioxidant activity when compared to methanolic extracts from organs of wild specimens. Finally, antimutagenic or mutagenic activity in wild plants and in vitro culture extracts was not detected by the Ames test.

Assessment of Genetic Stability of Micropropagated Bambusa balcooa Roxb. using RAPD Marker

Bambusa balcooa Roxb. was in vitro propagated by optimizing protocol using nodal segment from secondary branches with 100% success in MS liquid media containing 100 mg/l Myo-inositol, 3% sucrose supplement with 4.4 - 26.64 μM BAP for shoot multiplication, and 2.69 - 32.26 μM NAA for root induction. The highest shoot multiplication (14.53 ± 0.33 folds), shoot length (5.9 ± 0.6 cm), shoot number per explants (4.0 ± 0.24), and rooting (89.3 ± 0.33%) was obtained in MS liquid media supplement with 13.32 μM BAP (shooting) and 26.88 μM NAA (rooting) and 1% aqueous leaf extract of Artemisia vulgaris L. (EAV). Twenty RAPD (Random amplified Polymorphic DNA) primers were used individually to amplify DNA of tissue culture-raised plants and the mother plant where 8 primers yielded monomorphic banding patterns with reproducible, clear, scorable bands (2.8 per primer) ranging from 250 to 1800 bp respectively which revealed the micropropagated plants of B. balcooa retained their genetic stability.
 Plant Tissue Cult. & Biotech. 31(1): 81-95, 2021 (June)

High-frequency shoot regeneration from flower bud derived callus of Gymnostachyum febrifugum Benth., an endemic medicinal plant to the Western Ghats

Gymnostachyum febrifugum Benth. is a small, scapigerous, rare and endemic medicinal herb indigenous to India, and belonging to the family Acanthaceae. This study reports an efficient protocol for high-frequency flower bud-derived callus induction and shoot organogenesis in G. febrifugum. Flower buds at 7 days before anthesis (dBA) were excised from the inflorescence and cultured on MS medium supplemented with various concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D; 2.275–9.1 µM) for callus induction. The optimum callus induction (78%) was obtained on MS medium supplemented with 6.825 µM 2,4-D. The calli when subcultured on MS medium supplemented with different concentrations of thidiazuron (TDZ; 2.275–11.375 µM) or 6-benzylaminopurine (BAP; 2.22–11.1 µM) alone or in combination with 1- naphthaleneacetic acid (NAA; 1.074–3.759 µM) induced shoots. The highest frequency (94%) and number of shoots (44.6 shoots/unit callus) were obtained on MS medium supplemented with 9.1 µM TDZ and 2.685 µM NAA. The optimum rooting frequency (95%) and number of roots (10.2) were observed on ½ MS medium supplemented with 14.76 µM indole-3-butyric acid (IBA). The rooted plantlets were acclimatized and transferred to soil with 94% success. A protocol for high-frequency shoot induction from flower bud derived calli has been standardized for G. febrifugum,an endemic medicinal herb to the Western Ghats.

Banana Cultivars Microshoot Induction and Plantlet Formation Using Cytokinin and Auxin

<p>Banana is a horticultural plant with very high potentials, which contains carbohydrates and vitamins that are useful in fulfilling people's food and nutritional needs. Hence, this study aims to produce superior banana seedlings and develop a protocol for their mass production using a plant <em>in vitro</em> culture technique. This was a two stage-experiment i.e. microshoot production and plantlet formation. The result showed that Gebyar cultivar produced more shoots than the Kepok Kuning<em> </em>cultivar, with an average of 4.25 microshoots explant<sup>-1</sup>. However, Kepok Kuning produced more leaves than Gebyar,<em> </em>with an average of 4.64 leaves plantlet<sup>-1</sup>. Banana shoots cultured on the media containing Indole-3-acetic acid (IAA) at a concentration of 2.5 µM produced the highest leaves number. Meanwhile, those cultured on the media containing 1-Naphthalenesacetic acid (NAA) at a concentration of 7.5 µM produced the highest roots number. A Murashige and Skoog (MS) medium supplemented with 6-Benzylaminopurine (BAP) up to 30 µM and the one supplemented with 7.5 μM of NAA are suitable for Kepok Kuning and Gebyar<em> </em>cultivars micropropagation with regard to microshoot induction and plantlet formation, respectively.</p>

Regeneration of Hemidesmus indicus (L.) R. Br. using in vitro nodes: an alternative method for efficient multiplication of shoots

In vivo nodes of Hemidesmus indicus (L.) R. Br. induced healthy multiple shoots with branching in our earlier studies and thus in the present study, potency of in vitro nodes to regenerate shoots was evaluated. In vitro nodes were excised from eight-week-old shoots and placed in Murashige and Skoog’s (MS) medium fortified with sucrose (3%) and different concentrations of 6-benzyladenine (BA) and kinetin (Kn). After eight weeks, optimum of 5.42 ± 0.36 shoots with 100% response were regenerated in medium supplemented with BA (10 µM) and Kn (5 µM). These healthy shoots were placed in full, half and quarter strengths of liquid MS medium fortified with sucrose (1%) and α-naphthaleneacetic acid (NAA, 1-25 µM) for rooting. Among all the strengths of MS medium, full strength MS medium having 8 µM NAA formed maximum of 3.42 ± 0.55 roots (91.67% response) within four weeks. The protocol is in continuation with earlier study and it was confirmed that a single in vivo nodal explant can regenerate around 385 healthy elongated shoots within 4 months, which will help in mass-propagation of the species.