Shoot Bud Induction Research Articles

The Establishment of a Highly Efficient In Vitro Regeneration System for Viburnum opulus L. ‘Roseum’

Viburnum opulus L. ‘Roseum’ is a highly valuable ornamental plant for landscaping, but it has a long propagation cycle and low propagation coefficient. In this study, stem segments with axillary buds from Viburnum opulus L. ‘Roseum’ were used as explants. We systematically analyzed the use of sodium hypochlorite for the sterilization of explants, as well as the effects of different plant growth regulator combinations and concentrations on shoot bud induction, shoot proliferation, the rooting of tissue-cultured shoots, and the transplanting of the tissue-cultured shoots. A complete rapid propagation technology system for Viburnum opulus L. ‘Roseum’ was established. The results showed that a disinfection method using 75% ethanol for 30 s and soaking in 5% sodium hypochlorite for 5 min was the most suitable for disinfecting the stem segments of Viburnum opulus L. ‘Roseum’, which showed low contamination and a 73.33% survival rate. The ideal medium for primary bud induction was WPM (Woody Plant Basal Medium) + 2.0 mg·L−1 6-benzylaminopurine (6-BA) + 0.15 mg·L−1 indole-3-butyric acid solution (IBA) + 25 g·L−1 sucrose. The optimal medium for shoot proliferation was WPM + 1.0 mg·L−1 6-BA + 0.15 mg·L−1 IBA + 25 g·L−1 sucrose, achieving an induction rate of 7.17. For the rooting of tissue-cultured shoots, the most suitable formulation was 1/2 WPM + 0.3 mg·L−1 naphthaleneacetic acid (NAA) + 0.3 mg·L−1 activated charcoal (AC) + 25 g·L−1 sucrose, which induced robust and developed root systems. This study provides a technical basis for the establishment of a fast propagation system for the industrial production of Viburnum opulus L. ‘Roseum’.

In vitro Germination and Mass Multiplication of an Endangered Medicinal Orchid Bulbophyllum crassipes Hook. f. from Bangladesh

An efficient procedure for mass multiplication of an epiphytic medicinal orchid from Bangladesh Bulbophyllum crassipes was established by in vitro seed culture. Seeds obtained from immature pods of B. crassipes were cultured on four different types of nutrient media viz. MS, PM, modified VW and B5. The maximum percentage of seed germination (90.75 ± 0.61) was recorded on MS where various plant growth regulators (PGRs) were used either individually or in combination for secondary protocorm development and multiple shoot buds (MSBs) induction. Secondary protocorm were developed from primary protocorm on MS with BAP, Kn and NAA. The highest number of secondary protocorms (21.07 ± 0.12) were obtain from primary protocorm in MS with 1.0 mg/l Kn and 0.5 mg/l NAA. Here 1.0 mg/l BAP and 0.5 mg/l NAA showed the best performance on plant height (3.64 ± 0.07 cm). Maximum multiple shoot buds (8.40 ± 0.26) per single shoot were developed on MS with 1.0 mg/l BAP and 0.5 mg/l NAA. For root induction single shoots were sub-cultured on ½ MS supplemented with NAA, IAA and IBA. Maximum root induction (6.50 ± 0.29) and its growth (4.11 ± 0.08 cm) was observed on ½ MS supplemented with 1.0 mg/l IAA. The well rooted plantlets were acclimatized and successfully transferred to pot containing coconut husk, brick pieces and charcoal at a ratio of 2:1:1 for further growth and development. Plant Tissue Cult. & Biotech. 34(2): 93-104, 2024 (December)

Micropropagation, micromorphological evaluation and genetic homogeneity validation of Cucumis collosus (Rottl.) Cogn.: A wild progenitor of melon

Cucumis collosus (Rottl.) Cogn., a wild ancestor of melon cultivars, is an underutilized cucurbit of arid horticulture having nutraceutical and therapeutic significance. C. collosus has great potential for enhancing the genetic base of cultivated melon and other cucurbits. This investigation is the first report on a robust and reliable micropropagation system for C. collosus (var. AHK-119) using nodal explants, followed by micromorphological and genetic stability assessment. More than 95% explants showed axillary shoot-bud induction on MS (Murashige and Skoog, 1962) medium containing 0.5 mg L−1 BAP and developed a maximum of 3.2 ± 0.6 shoots measuring 3.2 ± 0.1 cm length. After evaluating the various parameters, the best shoot multiplication (Shoot Number 34.9 ± 1.7, Shoot Length 6.7 ± 0.9 cm) was achieved on MS medium having half-strength of nitrates (825 mg L−1 NH4NO3 and 950 mg L−1 KNO3) + 0.25 mg L−1 6-benzylaminopurine (BAP) + 0.1 mg L−1 Kinetin (Kin) + 0.1 mg L−1 Indole-3-acetic acid (IAA) + additives (50 mg L−1 ascorbic acid and 25 mg L−1 each of adenine sulfate, L-arginine and citric acid), after 4 weeks. Micro-shoots were rooted using three approaches: In Vitro Continuous Treatment (IVCT), In Vitro Pulse-Treatment (IVPT) and Ex Vitro Pulse Treatment (EVPT). Of these, EVPT approach proved to be most successful in terms of rooting response (80%), root number (6.4 ± 0.9) and root length (5.7 ± 0.7 cm). The foliar micro-morphology analysis of in vitro leaves (IVL), ex vitro leaves (EVL) and mother plant leaves (MPL) demonstrated notable adaptive alterations during the transitional stages and produced structurally stable plants similar to the mother plant. Furthermore, the validation of genetic homogeneity using Start Codon Targeted (SCoT) and Inter Simple Sequence Repeats (ISSR) markers revealed monomorphic banding patterns among the micropropagated and the mother plant. The discussed method can be applied for large-scale commercial production of C. collosus for arid horticulture, urban/vertical farming, breeding purposes and improvement of other cucurbits.

In vitro Propagation of Leucas biflora (Vahl) Sm. - A Vulnerable Medicinal Herb

A rapidly well-developed in vitro regeneration system has been established for Leucas biflora using nodal segments as explants and MS medium supplemented with various concentrations and combinations of plant growth regulators (PGRS). MS medium containing 2.0 mg/l BAP and 0.5 mg/l NAA showed the highest response (95.99%) in induction of shoot buds. This media combination also produced the maximum number (11.20 ± 0.49) of multiple shoot bud per explant after 3 subcultures at an interval of 14-days. Microshoots showed the maximum elongation (4.70 ± 0.07 cm) in the same medium along with subcultures. These elongated shoots were further grown on rooting media, producing vigorous and healthy root systems. The best response (95.99 ± 1.63%) for rooting was obtained when half-strength MS medium was supplemented with 0.5 mg/l IAA + 0.5 mg/l IBA in 21 days of culture. In this combination, the average number and length of roots were 5.20 ± 0.33 and 4.80 ± 0.30 cm, respectively. The seedlings were removed from culture tubes and transplanted into the pots via several stages of acclimatization. Finally, 98% of the seedlings were found to survive under natural condition. Plant Tissue Cult. & Biotech. 34(1): 49-54, 2024 (June)

Influence of growth regulators on callus induction and plant regeneration from anthers of Tagetes spp.

Marigold (Tagetes spp.) belongs to family Asteraceae, native to Mexico, is one of the most important flower crop grown commercially in different parts of India. The application of haploid techniques as anther culture can considerably accelerate marigold breeding programmes by providing homozygous doubled haploid (DH) lines for F1 hybrid seed production. Therefore, the objective of the present investigation was to study the effect of growth regulators for in vitro regeneration in both African marigold and French marigold genotypes viz., Pusa Basanti Gainda and Pusa Arpita, respectively for haploid induction. Among the different treatments tested for callus induction, anthers cultured on MS medium supplemented with 1.0 mg/l BAP + 1.0 mg/l 2,4-D + 45 g/l sucrose significantly induced highest (38.74%) callus in the shortest period of time (16 days). Among two genotypes tested, embryogenic callus induction (27.70%) was significantly higher in Pusa Arpita over Pusa Basanti Gainda (23.37%). Among the different regeneration treatments, the highest adventitious shoot bud induction (13.32%), maximum (1.09) number of buds/callus in shortest duration (19.47 days) was observed in anther derived callus cultured on MS medium supplemented with 2.0 mg/l BAP + 0.5mg/l NAA + 30 g/l sucrose. Among two genotypes evaluated for shoot bud induction, maximum (3.81%) regeneration was recorded in Pusa Arpita over Pusa Basanti Gainda (2.90%). In French genotype, the whole regeneration process was completed in 30 days whereas in African genotype it took nearly 40 days. This rapid regeneration system from anthers is highly useful in inducing haploids and di-haploids in African and French marigold lines, respectively.

In vitro regeneration of triploid from mature endosperm culture of Passiflora edulis "Mantianxing".

The regeneration of shoots from endosperm tissue is a highly effective method to obtain triploid plants. In this study, we elucidated the establishment of an in vitro regeneration system from endosperm culture for the production of Passiflora edulis "Mantianxing." The highest callus induction rate (83.33%) was obtained on the media supplemented with 1.0mg/L TDZ. Meanwhile, the MS medium containing 1.0mg/L 6-BA and 0.4mg/L IBA gave the optimum 75% shoot bud induction. Chromosome analysis revealed that the chromosomal count of P. edulis "Mantianxing" regenerated from endosperm tissues was 27 (2n=3x=27), which indicated that shoots regenerated from endosperm tissues were triploids. Triploid P. edulis had more drought resistance than diploid plants. Our study provided a method for breeding of passion fruit by means of a stable and reproducible regeneration system from endosperm culture, leading to the generation of triploid plants.

Somatic Embryogenesis and Direct Shoot Bud Formation from in vitro Root Segments of Garlic (Allium sativum L.)

In vitro techniques are unconventionally used for garlic improvement. In garlic tissue culture, numerous roots are produced in vitro. Segments (1 cm) of those in vitro roots were cultivated on MS medium with 5.0 μM 2, 4-D for callus initiation followed by transfer to 0.5 μM 2, 4-D for somatic embryogenesis. Root segments had 51.67% callus and somatic embryo formation. Plantlet regeneration was obtained from 90% calli after being transferred to MS medium supplemented with 5.0 μM kinetin. Within 6 months, 54.8 plants per root segment and 42.2 plants per root tip explant were found. MS media supplemented with 1.0 or 5.0 μM NAA in combination with 10.0 or 50.0 μM BA were used for direct shoot bud induction from root segments. The highest percentage (23.33 %) of direct shoot bud regeneration was found in 5.0 μM NAA with 50.0 μM BA within 2 months. The number of shoots per explant was varied from 1-81 with the highest average of 15.75 shoots per explant. The shoots produced bulblets upon transfer to a medium containing higher amount of sucrose (12%). The largest bulblets weighed 356.57 g in the medium with 5.0 μM NAA and 50.0 μM BA. In both pathways, regeneration occurred from only the apical (distal) end of the root segments. The protocols are promising for continuous regeneration of propagules and genetic transformation study for improvement of garlic. Plant Tissue Cult. & Biotech. 33(2): 135-142, 2023 (December)

Micropropagation and cytological studies of Aole vera Linn

Aloe vera Linn. is an essential medicinal plant. In this present research work, a protocol of in vitro regeneration and karyomorphological analysis of Aloe vera was developed using different concentrations and compositions of media. Shoot apices of field-grown plants were used as explant and aseptically cultured on Murashige and Skoog (MS) medium fortified with different concentrations and combinations of auxins (IAA and NAA) and cytokinins (BAP and Kn). The highest number of multiple shoot buds (4.36 ± 0.07) was obtained from MS + 2.0 mg/l BAP + 1.0 mg/l IAA and induced shoot buds underwent rapid elongation (4.24 ± 0.06 cm) on the same medium composition. Half strength MS media with 2.0 mg/l IBA was suitable for induction and proliferation (6.31 ± 0.05) of roots and 95% of plantlets were acclimatized to field conditions successfully. Somatic chromosome numbers of mother and in vitro grown plants were confirmed to be 2n = 14. Chromosome length ranged from 4.28 - 13.74 µm in the naturally grown plants and 4.46 - 14.1 µm for in vitro grown plants. The total form percent (TF%) of mother and in vitro grown plants was 41.69% and 42.23%, respectively. The karyotype formula of in vivo grown plants was 2n = 14 = 4Lsm + 6Mm + 4Sm, whereas that of the micropropagated plants was 2n = 14 = 4Lsm + 4Mm + 6Sm. The frequency of the chromosome having arm more than 2:1 was 0.08 for mother plants and 0.15 for in vitro grown plants. Therefore, the karyotype of both plants falls into the 2B symmetrical type based on Stebbins classification (1971).

In vitro study of virus new strain JLCuGV infected nodal explants and production of planting material through meristem culture from virus-infected Jatropha curcas plants

Abstract Jatropha curcas is an important biodiesel plant as its seed contains 27–40% oil. The virus infection causes adverse effects on plant growth, and yield. The present study was carried out to develop plantlets from virus-infected plants using meristematic cultures. Cultures were also raised using nodal explant to figure out culture loss caused by the new strain of Jatropha leaf curl Gujarat virus (JLCuGV). Poor (48.3 ± 15.1 %) shoot bud induction was noted in virus-infected explants on MS medium supplemented with 6-benzylaminopurine (BAP) and Indole-3-acetic acid (IAA). Severe culture loss was noted upon subsequent sub-cultures of nodal explants. Apical meristem (MC) developed the highest (86.3 ± 12.5 %) shoot bud induction on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L Thidiazuron (TDZ). Best results in shoot proliferation and elongation were achieved on 1.0 mg/L BAP, 1.5 mg/L IAA and 0.5 mg/L Gibberellic acid (GA3) containing medium with 9.9 ± 1.7 number of shoot buds of 5.2 ± 0.5 cm shoot length. It was interesting to note that the rooting percentage was not affected by virus infection. Shoots obtained from virus-infected and meristem cultures rooted well without any significant difference in rooting percentage. It was known by the study that the generation of planting material from virus-infected plants was possible with meristem explants but recalcitrant with nodal explants.

Rapid and Efficient Regeneration of Populus ussuriensis Kom. from Root Explants through Direct De Novo Shoot Organogenesis

Populus ussuriensis is an important tree species with high economic and ecologic values. However, traditional sexual propagation is time-consuming and inefficient, challenging afforestation and wood production using P. ussuriensis, and requires a rapid and efficient regeneration system. The present study established a rapid, efficient, and stable shoot regeneration method from root explants in P. ussuriensis using several plant growth regulators. Most shoot buds (15.2 per explant) were induced at high efficiency under WPM medium supplemented with 221.98 μM 6-BA, 147.61 μM IBA, and 4.54 μM TDZ within two weeks. The shoot buds were further multiplicated and elongated under WPM medium supplemented with 221.98 μM 6-BA, 147.61 μM IBA, and 57.74 μM GA3 for four weeks. The average number and efficiency of elongation of multiplication and elongation for induced shoot buds were 75.2 and 78%, respectively. All the shoots were rooted within a week and none of them showed abnormality in rooting. The time spent for the entire regeneration of this direct shoot organogenesis was seven weeks, much shorter than conventional indirect organogenesis with the callus induction phase, and no abnormal growth was observed. This novel regeneration system will not only promote the massive propagation, but also accelerate the genetic engineering studies for trait improvement of P. ussuriensis species.

Effect of Plant Growth Regulators on in vitro Morphogenic Response of Gliricidia [Gliricidia sepium (Jacq.) Steud.

Background: The present investigation was carried out with the objective of optimization of rapid micropropagation protocol and to evaluate effect of different concentrations of plant growth regulators added singly or in combinations on in vitro culture of Gliricidia sepium. Methods: Explants (Soft nodal stem segments) were inoculated on MS medium containing varying concentrations of cytokinins and auxins either singly or in combinations. The cultures were incubated at 25±2°C for 14: 10 hour’s photoperiod with a light intensity of 3000-3500 lux. Result: Maximum number of shoot bud induction obtained when MS medium was supplemented with 0.5 mg/l BAP with 100 per cent frequency. Highest frequency (100%) of rooting was observed at 0.5 mg/l of IAA. 2.0 mg/l NAA showed highest number of roots/explant but the roots were thick. For shoot bud induction, BAP was found to be most effective among all the plant growth regulators. For root induction, both NAA and IAA were found to be effective but IAA exhibited higher frequency of rooting as compared to NAA.

Comparative study of micropropagated plants of Grand Naine banana during in vitro regeneration and ex vitro acclimatization

An improved micropropagation protocol was established from field grown sword suckers of Banana Grand Naine (Musa acuminata AAA) by evaluating the mircopropagules combating reactive oxygen species (ROS) during acclimatization. Optimization of surface sterilization of sprouted apical meristematic buds and pre-culture on Shoot Bud Induction Medium [MS + 4.0 mg l −1 BA under dark conditions for 3 weeks] resulted ideal number of multiple shoot clusters (12.20 ± 0.30). Subculture of these shoot clusters on Shoot Bud Multiplication Medium (SBMM) fortified with 2 mg l −1 BA and 20 mg l −1 AdS induced highest shoot buds (15.00 ± 0.75) in 3 weeks and showed consistent till seven passages. The individual in vitro shoots upon transferring onto MS + 2 mg l −1 KN +1.0 mg l −1 IBA + activated charcoal (3 g l −1) resulted in elongated shoots and in vitro rooting after 3 weeks at a photoperiod of 16 h light and 8 h dark conditions. Evaluation of antioxidant defense status in micropropagated plantlets during acclimatization revealed altered levels of MDA, chlorophyll, catalase, ascorbate peroxidase and glutathione reductase. Our study throws an insight into decisive/differential role of cytokinins (Kinetin) in combating oxidative stress during acclimatization of in vitro regenerated plantlets. The present investigation facilitates the massive propagation (90% survival rate) of healthy and quality in vitro plantlets of banana.

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.

Validation of meta-Topolin in organogenesis, improved morpho-physio-chemical responses, and clonal fidelity analysis in Dioscorea pentaphylla L. – an underutilized yam species

The present investigation describes the efficacy of aromatic and synthetic cytokinins, which influence the direct organogenesis, biochemical responses, and ex vitro and in vivo performance of the in vitro propagated plantlets of Dioscorea pentaphylla L., an underrated famine tuber crop. The axillary shoot bud induction was achieved on the Murashige and Skoog's (MS) medium supplemented with 1.0 mg L−1 meta Topolin (mT) (96.9%) and 1.0 mg L−1 Thidiazuron (TDZ) (82.4%). The sprouted shoots were proliferated using 0.5 mg L−1 mT combined with 0.1 mg L−1 indole-3-acetic acid (IAA) with the maximum average number of 43.0 ± 1.29 shoots (7.2 ± 0.55 cm length). The shoots proliferated on mT and IAA combination demonstrated the presence of higher concentrations of carbohydrates and photosynthetic pigment contents in the leaves as compared with TDZ and IAA derived leaves. The healthy micro-shoots were rooted on a half-strength MS medium containing 1.0 mg L−1 indole-3-butyric acid (IBA) with the development of 12.6 ± 0.38 roots (5.9 ± 0.22 cm length). The plantlets were acclimatized in a greenhouse and showed 100% survival under in vivo conditions. The clonal fidelity of the regenerants was evaluated for the first time using Start codon targeted (SCoT) markers. Thirteen primers amplified 62 well-resolved bands and ranged from 1-8 bands. There were no somaclonal variations detected among the regenerants of D. pentaphylla with the mother plant.

Development of an Efficient In vitro Regeneration Protocol for Chrysanthemum (Chrysanthemum morifolium Ramat)

An efficient and rapid in vitro regeneration protocol was developed for chrysanthemum (Chrysanthemum morifolium Ramat) using two local varieties of Bangladesh namely, BARI Chrysanthemum-2 (BARI Chry-2) and local yellow (Y). MS medium supplemented with nine different concentrations and combinations of BAP and IAA was employed to optimize regeneration protocol using young in vitro derived leaf explants. Direct organogenesis was observed from the leaf explants on MS medium supplemented with 0.5 mg/l BAP and 2.0 mg/l IAA (T6) for both the varieties. This treatment (T6) induced shoot buds directly on the adaxial surface of the leaf providing the highest regeneration percentage (90% for BARI Chry-2 and 94.73% for Y), the highest number of shoot/explant (7.6 for BARI Chry-2 and 8.6 for Y) and maximum length of the shoot after six weeks (3 cm for BARI Chry-2 and 2.9 cm for Y) of culture. Explants with initially regenerated shoots were subculture on hormone free MS medium for shoot elongation after 4 weeks of their inoculation. During elongation of shoots, 90-95% of the regenerated shoots produced roots spontaneously in hormone free MS medium within 7-8 weeks of their inoculation. Rooted plantlets were transplanted to the field following hardening where 100% plantlets were survived and produced flower without any variation. Plant Tissue Cult. & Biotech. 31(2): 161-171, 2021 (December)

De Novo Shoot Bud Induction from the Catharanthus roseus Leaf Explants and Agrobacterium tumefaciens-Mediated Technique to Raise Transgenic Plants.

The regeneration of a whole plant from a single cell or organ explant was a valuable task for plant biotechnology. However, important medicinal plants such as Catharanthus roseus have shown recalcitrance to regeneration protocols, thus limiting investigations on MIA metabolism and metabolic engineering in this plant system. In this chapter, successful regeneration protocols were detailed for Catharanthus roseus, either by direct shoot bud induction from leaf explants and Agrobacterium-mediated genetic transformation.

Multiple shoot induction and regeneration of Vanilla borneensis Rolfe - a critically endangered orchid of Assam, India

In the present investigation, a micropropagation protocol has been developed for Vanilla borneensis Rolfe – a critically endangered orchid through multiple shoot regeneration. Through in vitro multiple shoot regeneration from both nodal and shoot tip explants, maximum (100%) shoot induction was observed. The minimum time required for shoot bud induction was observed from the shoot tip (5–7 days) on medium supplemented with BAP (4.44 mM) + KIN (2.32 mM) as compared to the nodal explants. Maximum multiple shoot regeneration was observed from nodal explants on the medium supplemented with BAP (4.44 mM) + TDZ (6.82 mM). However, maximum shoot length was observed on the medium supplemented with BAP (4.44 mM) + 15% CW and the number of nodes (5.27±0.33) per shoot after 90 days. Maximum (80-100%) of root initiation was observed in almost all the concentrations of NAA. The shortest time of root initiation was found on the medium supplemented with NAA (5.37 mM). Further, acclimatization period was found to be 15 days with 70% acclimatization while 60% of survivability was observed in the field condition. This efficient micropropagation method of V. borneensis could be successfully used for mass propagation as well as conservation of the critically endangered wild orchid.

Micropropagation of Rare Scutellaria havanensis Jacq. and Preliminary Studies on Antioxidant Capacity and Anti-Cancer Potential.

We report the development of in vitro propagation protocols through an adventitious shoot induction pathway for a rare and medicinal Scutellaria havanensis. In vitro propagation studies using nodal explants showed MS medium supplemented with 10 µM 6-Benzylaminopurine induced the highest number of adventitious shoots in a time-dependent manner. A ten-day incubation was optimum for shoot bud induction as longer exposures resulted in hyperhydricity of the explants and shoots induced. We also report preliminary evidence of Agrobacterium tumefaciens EHA105-mediated gene transfer transiently expressing the green fluorescent protein in this species. Transformation studies exhibited amenability of various explant tissues, internode being the most receptive. As the plant has medicinal value, research was carried out to evaluate its potential antioxidant capacity and the efficacy of methanolic leaf extracts in curbing the viability of human colorectal cancer cell line HCT116. Comparative total polyphenol and flavonoid content measurement of fresh and air-dried leaf extract revealed that the fresh leaf extracts contain higher total polyphenol and flavonoid content. The HCT 116 cell viability was assessed by colorimetric assay using a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide, showed a steady growth inhibition after 24 h of incubation. Scanning electron microscopy of leaf surface revealed a high density of glandular and non-glandular trichomes. This research provides a basis for the conservation of this rare plant and future phytochemical screening and clinical research.

Development of high frequency cost-effective micropropagation protocol for Juncus rigidus using liquid culture medium and extraction of cellulose from their in vitro shoots - An important rush

Juncus rigidus is important halophyte with high cellulose content, but no studies has been made on micropropagation of the plant and cellulose extraction from in vitro shoots. To optimize an effective system for shoot multiplication, explants were grown in agar medium, liquid static culture, and bioreactor. Best shoot bud induction was obtained in liquid MS medium supplemented with2.27 μM thidiazuron (TDZ) over 0.46–4.65 μM Kinetin (KN) and 2.22–6.66 μM 6-benzyl aminopurine (BAP). Maximum number of shoot bud (36 ± 1.3) were achieved in static liquid MS medium supplemented with 2.22 μM BAP and 0.57 μM indole acetic acid (IAA). Upon subsequent subcultures >80 shoot buds were achieved in static liquid MS medium supplemented with 1.11 μM BAP, 0.57 μM IAA, and 0.46 μMKN within 30 days of culture. Being halophytic plant NaCl did not have any significant effect on shoot proliferation. Elongated shoots cultured in MS medium supplemented with 0.44 μM BAP, and 0.57 μM IAA developed 97% rooting in filter paper boat culture vessel. Sapling production cost in liquid culture medium was 90% less as compared to solid medium. Cellulose was extracted from the in vitro shoots and 17.64% cellulose achieved from liquid medium cultured shoots. For the first time micro-shoots are tested for in vitro cellulose production. • Maximum shoot bud (36 ± 1.2) induction was achieved in static liquid medium. • More than 80 shoot buds generated in static liquid MS medium supplemented with BAP, IAA, and KN. • 33.4 multiplication rate was recorded in static liquid medium. • 17.64 % cellulose extracted form in vitro shoots of J. rigidus. • In vitro plant based cellulose synthesis is important with future industrial application and bioengineering.

Exogenous application of 6-benzyladenine and spermine improves shoot bud induction of shoot apex of Terminalia bellerica Roxb.

Shoot regeneration was optimized from shoot apex explants of mature tree species Terminalia bellerica using 6-benzyl adenine (BA) and spermine (Spm). The maximum shoot bud regeneration response (100%) was observed on BA supplemented Murashige and Skoog (MS) medium. However, MS medium with 2.22 μM BA and 9.90 μM Spm induced an average of 21 micro shoots per shoot apex and averaged 2.4 cm shoot length after 4 weeks of culture. Of four combinations tested, 2.22 μM BA + 1.50 μM gibberellic acid (GA3) was found to be most responsive for shoot bud proliferation, followed by intermediate response on BA (4.44 μM), BA (2.22 μM) and further lower on combination of 2.22 μM BA, 1.50 μM GA3 and 9.90 μM Spm. Micro shoots production reached to 100%, with maximum number of 32 micro shoot buds per explant cultured on MS medium with 2.22 μM BA and 1.50 μM GA3 after an interval of 8 weeks. Between auxin combinations as indole-3-acetic acid (IAA)/indole-3-butyric acid (IBA), IAA/naphthalene acetic acid (NAA) or IBA/NAA, the root induction was highest (94%) on the latter medium added with 2.46 μM IBA and 2.69 μM NAA producing 11.8 healthy roots per shoot with an averaged root length of 1.8 cm within 15–30 days of culture. Successful acclimatization of plantlets in the garden soil resulted to 90% healthy plants with no differences observed in the morphological characteristics of the growing shoots.