Shoot Buds Research Articles

Micropropagation of belarusian cultivars of blue honeysuckle (Lonicera caerulea L. var. kamtschatica)

For blue honeysuckle cultivars of Belarusian selection (Zinri and Sinyavokaya), a method of micropropagation for the production of high-quality planting material is proposed. Initiation of in vitro culture: in the period of intensive growth of shoots – the first decade of June. Explants were apical points isolated from the apical and axillary buds of green shoots. The nutrient medium was WPM supplemented with 6-BA at a concentration of 1.0 mg/l. Micropropagation stage: MS medium containing double strength iron chelate with 1.5 mg/l of 6-BA. In vitro rhizogenesis: in MS medium the concentration of macroand microsalts and iron chelate was reduced to half strength, the sucrose concentration was reduced to 20 g/l, IBA – 1.5–2.0 mg/l). Ex vitro adaptation: substrate – perlite. It is possible to reduce the cost of obtaining the planting material by eliminating in vitro rooting stage from micropropagation scheme. Simultaneous direct ex vitro rooting and adaptation of microplants should be carried out on the substrate of Sphagnum L. moss with a surface layer (0.5 cm) of peat. Long-term storage (up to 12 months) at low positive temperatures (+3–4 °C) in refrigerator should be carried out at rooting stage (in MS medium the concentration of macro- and microsalts and iron chelate was reduced to half strength, the sucrose concentration was reduced to 20 g/l, IBA – 1.0 mg/l).

Optimizing in vitro nutrient and ex vitro soil mediums-driven responses for multiplication, rooting, and acclimatization of pineapple

A comprehensive study was carried out on in vitro multiplication and rooting using the medium enriched with different plant growth regulators and acclimatization of pineapple cv. ‘Smooth Cayenne’ using different soil growing substrates. The significantly highest shoot buds (Avg. 16.7) were obtained on the medium comprising 2.0 mg L−1 BA (6-Benzylaminopurine). Results showed that 1.0 mg L−1 IBA (Indole-3-butyric acid) increased the thickness and length of white adventitious roots and resulted in a significantly highest number of roots (Avg. 8) and root length (6.15 cm). Plantlets with healthy, multiple roots were transplanted in several soil combinations of river silt, bolhari (yellow sand), and peat moss. However, the significantly highest survival (100%) of plantlets in the greenhouse was obtained on the soil medium containing only peat moss. Furthermore, soil mixtures of bolhari and peat moss (1:1) and river silt alone exhibited 98.9% and 95.1% survivability of plantlets, which was also considered equally significant (at 5% probability level). The in vitro and ex vitro protocols optimized in the current study can be applied commercially for pineapple crop worldwide.

Influences of Various Factors on Inducing Adventitious Shoot Regeneration From Stem Explants of Cassia Mimosoides Linn.

In the present study, an efficient plant regeneration method was established in Cassia mimosoides by using stem explants. Low regeneration frequency with poor quality advantageous shoot buds were obtained when the explants were grown only Kinetin (KN) or thidiazeuron (TDZ) enriched media. For improving the regeneration efficiency and production of good quality adventitious buds, Cu2+, Mg2+, Fe2+ and Ag+ were supplemented in the regeneration medium at different concentrations. The highest (63.96%) adventitious bud regeneration rate, the maximum number of buds (5.39) per explant and the longest regenerated buds (3.49 cm) were obtained by adding 26 mg/l Ag+ in the regeneration medium. In order to improve the rooting in shoot buds, diethyl aminoethyl hexanoate (DA-6) at different concentrations was added into rooting medium. The optimal rooting was obtained by supplementing 0.6 mg/l DA-6. The highest rooting rate (90.23%), the highest number of roots per bud (6.08) and the longest roots (4.26 cm) were developed within 20 days of culture in this medium. By applying this method culture cycle for obtaining complete regenerated plantlets was significantly shortened (in 60 days), Additional culture for proliferation and elongation of regenerated adventitious buds was not needed. Therefore, the method established in the present study was helpful to improve the efficiency of plant regeneration from stem explants in C. mimosoides. Besides the establishment of this culture method could lay a foundation for the future large-scale cultivation and genetic transformation of C. mimosoides. Bangladesh J. Bot. 51(4): 873-882, 2022 (December) Special

In vitro bioactive metabolite production and plant regeneration of medicinal plant Angelica sinensis

The root of Angelica sinensis (Oliv.) Diels is a traditional Chinese medicine (TCM) widely used for the cardio-cerebrovascular, anti-inflammatory, and anti-cancer agents, etc. Currently, the root yield is mainly limited by narrow geographical distribution, early bolting and flowering (EBF), continuous cropping obstacle, and germplasm degeneration. While two ancillary strategies to produce bioactive metabolites by bioreactor and innovate new germplasms by genome editing have not been conducted. Here is reported embryogenic callus induction, histomorphological structure identification, suspension cell culture, bioactive metabolite determination, shoot bud and root differentiation, and plantlet acclimatization at different treatments such as explants, hormones, and culture conditions. The embryogenic calli can be effectively induced (99.0%) from the root of aseptic seedlings by a combination of 2.0 mg/L IBA and 0.2 mg/L KT, and embryogenic cells including: three-cell, globular-, heart-, torpedo-, and cotyledon-shaped embryos were observed. The maximum growth rate, bioactive metabolites contents (i.e., ferulic acid, soluble sugar, flavonoids, and phenolic), and antioxidant capacities were observed at 9–12 d with inoculum density 40 g/L, 22 ℃ and 16/8 h light/dark. Robust plantlets can be obtained within 180 d after successive shoot bud and root differentiation as well as plantlet acclimation. Based on the strategies for the satisfaction of root demand, an effective and complete scheme is proposed for producing bioactive metabolites and regenerating plant of A. sinensis.

In vitro plantlet regeneration from cotyledon explants of Capsicum annuum L

An efficient In vitro plant regeneration was established in Capsicum annuum L. from cotyledon explants. The best performance i.e., 80% of the explants responded for multiple shoot bud proliferation on MS medium containing 10 mg/l Zeatin. The maximum number of buds per explants was 50.28 ± 2.69 and the bud length of 0.5 ± 0.22 cm was obtained in this medium after 30 days. The average number of 80.32 ± 0.16 multiple shoot bud and the average shoot bud length of 1.0 ± 0.19 cm was attained on the same medium after 90 days. These shoot buds on transfer to MS + 10 mg/l Zeatin + 2.0 mg/l GA3 yielded 6.3 ± 0.35 number of multiple shoots with an average shoot length of 4.6 ± 0.62 cm after 30 days. Shoots rooted well (80% of induced shoots) on half strength MS medium supplemented with 0.5 mg/l IBA. The average number of roots per shoot was 8.2 ± 0.79 and the average root length of 4.5 ± 0.58 cm was recorded. About 80% of In vitro derived plantlets were survived under open field conditions.

Ploidy Status, Nuclear DNA Content and Start Codon Targeted (SCoT) Genetic Homogeneity Assessment in Digitalis purpurea L., Regenerated In Vitro.

Digitalis purpurea L. is a therapeutically important plant that synthesizes important cardiotonics such as digitoxin and digoxin. The present work reports a detailed and efficient propagation protocol for D. purpurea by optimizing various PGR concentrations in Murashige and Skoog (MS) medium. The genetic homogeneity of in vitro regenerants was assessed by the flow cytometric method (FCM) and Start Codon Targeted (SCoT) marker technique. Firstly, the seeds inoculated in full MS medium added with 0.5 mg/L GA3 produced seedlings. Different parts such as hypocotyl, nodes, leaves and apical shoots were used as explants. The compact calli were obtained on BAP alone or in combinations with 2, 4-D/NAA. The hypocotyl-derived callus induced somatic embryos which proliferated and germinated best in 0.75 mg/L BAP-fortified MS medium. Scanning electron microscopic (SEM) images confirmed the presence of various developmental stages of somatic embryos. Shoot regeneration was obtained in which BAP at 1.0 mg/L and 2.0 mg/L BAP + 0.5 mg/L 2,4-D proved to be the best treatments of PGRs in inducing direct and indirect shoot buds. The regenerated shoots showed the highest rooting percentage (87.5%) with 24.7 ± 1.9 numbers of roots/shoot in 1.0 mg/L IBA augmented medium. The rooted plantlets were acclimatized in a greenhouse at a survival rate of 85-90%. The genome size and the 2C nuclear DNA content of field-grown, somatic embryo-regenerated and organogenic-derived plants were estimated and noted to be 3.1, 3.2 and 3.0 picogram (pg), respectively; there is no alteration in ploidy status and the DNA content, validating genetic uniformity. Six SCoT primers unveiled 94.3%-95.13% monomorphic bands across all the plant samples analyzed, further indicating genetic stability among in vitro clones and mother plants. This study describes for the first time successful induction of somatic embryos from hypocotyl callus; and flow cytometry and SCoT marker confirmed the genetic homogeneity of regenerated plants.

Morphological and Anatomical Comparison between Tetraploid Stevia rebaudiana (Bertoni) Bertoni and its Parental Diploid

Stevia rebaudiana (Bertoni) Bertoni is a perennial herbaceous plant that produces natural low glycemic-index sweeteners alternative to sugar cane. The genetic improvement of Stevia needs to be investigated to increase its productivity. Although this plant has a narrow genetic diversity, genetic improvement through polyploidization may succeed. Meanwhile, genotypic characters of tetraploid Stevia were previously reported. This study then observed the phenotypic characters of the tetraploid plants, which aimed to evaluate the morphological and anatomical characteristics of tetraploid Stevia clones compared to its wild-type parental diploid plant. Three tetraploids and one diploid clone were grown in the field for 12 weeks. The results of qualitative descriptive characters showed that the tetraploid clones B60.3H8, P1T22 and P3T5 had habitus type, shoot bud shape, leaf margin, leaf venation, shape, and type of stem, type of flower, and seed similar to the diploid parental clone. However, the quantitative characters showed that the tetraploid clones had leaf size, stem diameter, root size, length of the single flower, length of the flower stalk, and length of fruit which were more extensive than those of the parental diploid clone. The leaf color of tetraploids was a darker green, and the range of initial flowering times was earlier. This finding gives more understanding of the phenotypic and anatomical characteristics of Stevia tetraploids compared to their parental diploid plant.

Regeneration competence of an endangered orchid, Vanda cristata Wall. ex Lindl. using leaf explants: A study in vitro

In the present paper, a protocol for plant regeneration was developed for micropropagation of an epiphytic orchid, Vanda cristata Wall. ex Lindl., by using whole leaf explants procured from five months old in vitro grown cultures. The regeneration competence of leaf explants was tested on two different nutrient media (M and KC) supplemented with auxins (IAA, IBA, NAA) and cytokinins (BAP, KN, TDZ) at different concentrations. The regeneration response was invariably initiated mostly at the bases of the leaf explants via shoot bud, protocorm like bodies (PLBs) and callus formation whereas leaf tips (apical side) showed very less response. KC medium supplemented with NAA(10.6 µM)+BAP(8.8 µM), acted synergistically; cent per cent explants initiated regeneration response via formation of callus, PLBs and shoot buds at the basal part of leaf explant and complete plantlets were obtained. Highest number of regenerants (6) was observed in this combination as compared to the control. The genetic stability analysis of in vitro regenerated plantlets of V. cristata was performed by using inter-simple sequence repeats (ISSR) primers; 6.67% polymorphism and 93.33% of monomorphism was calculated using five ISSR primers, that showed a high degree of genetic stability to obtain true-to-type plantlets. The protocol developed in this study may be useful for conserving V. cristata including other orchid species using different biotechnological approaches.

Post-Spring Frost Canopy Recovery, Vine Balance, and Fruit Composition in cv. Barbera Grapevines

Background and Aims. In viticulture, one of the effects of warming trends is the advance of budburst date and the consequent increased risk of spring frost-related damage. In 2021, severe frost events affected a large fraction of European viticulture. In a cv. Barbera vineyard, located in NW Italy, primary bud shoots (PBS), secondary bud shoots (SBS), and suckers (SK) were tagged after the occurrence of freezing temperatures in spring. The goal of the study was to clarify if SBS could partially restore yield loss and analyze their contribution to fruit composition. Methods and Results. The number of developing SBS and SK correlated with the number of killed PBS. While PBS bore 1.44 inflorescences per shoot, SBS had much lower fertility (0.4), with SK at intermediate levels (0.85). The vine yield was 40% of the previous season, with SBS bunches contributing just 17% of the total. SBS produced smaller and looser bunches, as compared with PBS (−28% mass and −27% compactness). At harvest, no difference was found in grape total soluble solids (TSS) among different shoot types. However, the TSS average was notably higher than that observed in the previous season (27.8°Brix vs. 23.3°Brix in 2020). Interestingly, while in PBS and SK, a direct correlation (linear and quadratic, respectively) existed between the leaf area to yield ratio (LA/Y) and grape TSS or total anthocyanins, this did not occur for SBS. Conclusions. In the case of spring frost damage, the number of PBS avoiding fatal injuries will drive agronomic results at harvest since SBS contribution to total yield is modest due to low shoot fruitfulness. The frost-induced increase in vine LA/Y leads to a dramatic rise in grape TSS and phenolics. Significance of the Study. When spring frosts kill a significant number of primary shoots, an altered grape composition at harvest should be expected due to changes in vine balance. Therefore, the vineyard management should be adjusted accordingly early in the season. Further studies are needed to test specific post-frost canopy management strategies ensuring yield, optimal fruit composition, and cane renewal.

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.

In vitro Propagation of Curcuma caesia Roxb. via Bud Culture Technique and ISSR Profiling of the Plantlets for Genetic Homogeneity

An in vitro propagation protocol has been developed for Curcuma caesia Roxb., an endangered medicinal plant by Foundation for Revitalisation of Local Health Traditions and Central Forest Department of India. The plant bears poorly germinated seeds and produces two-storage organs: rhizomes and multiple root tubers. Only rhizomes have medicinal-economic values. They serve as propagules too, which results in a shortage of planting material. Therefore, a complete one-year production cycle of C. caesia has been standardized through in vitro propagation including explants establishment (one month), subculture cycles (seven months), rooting (one month) followed by primary hardening (one month) and secondary hardening (two months). Dormant shoot buds on rhizome served as explants for culture initiation on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA) and kinetin (KIN) in combination with citric acid (CA), adenine sulfate (AdS) and indole- 3-acetic acid (IAA). Maximum bud break (70%) was obtained on MS with 8 mg L-1 BA, 8 mg L-1 KIN, 100 mg L-1 CA, 200 mg L-1 AdS and 2 mg L-1 IAA (standard medium). Shoot production potential continued on this medium during the subsequent seven-month-long subculture cycle. The in vitro raised shoots rooted best on ½-strength MS containing 1 mg L-1 indole-3-butyric acid. Plantlet survival rate was >95% after acclimatization. The genetic homogeneity of plantlets with the mother plant was analyzed using Inter Simple Sequence Repeats which generated a monomorphic banding pattern to confirm the uniformity of in vitro raised plantlets of C. caesia.

MINI BODY1, encoding a MATE/DTX family transporter, affects plant architecture in mungbean (Vigna radiata L.).

It has been shown that multidrug and toxic compound extrusion/detoxification (MATE/DTX) family transporters are involved in the regulation of plant development and stress response. Here, we characterized the mini body1 (mib1) mutants in mungbean, which gave rise to increased branches, pentafoliate compound leaves, and shortened pods. Map-based cloning revealed that MIB1 encoded a MATE/DTX family protein in mungbean. qRT-PCR analysis showed that MIB1 was expressed in all tissues of mungbean, with the highest expression level in the young inflorescence. Complementation assays in Escherichia coli revealed that MIB1 potentially acted as a MATE/DTX transporter in mungbean. It was found that overexpression of the MIB1 gene partially rescued the shortened pod phenotype of the Arabidopsis dtx54 mutant. Transcriptomic analysis of the shoot buds and young pods revealed that the expression levels of several genes involved in the phytohormone pathway and developmental regulators were altered in the mib1 mutants. Our results suggested that MIB1 plays a key role in the control of plant architecture establishment in mungbean.

High-frequency Shoot Multiplication and In Vitro Plantlet Regeneration from Shoot Bud Explants in Curcuma zedoaria Roscoe

ABSTRACT A rapid and efficient micropropagation protocol was developed for Curcuma zedoaria, using shoot buds as explants. Explants established on basal MS medium were transferred to 12 different medium compositions with different combinations and concentrations of plant growth hormones 6-benzylaminopurine (BAP), kinetin (Kn), and thidiazuron (TDZ). The best shoot multiplication was recorded in MS medium supplemented with BAP (2.0 mg L−1) and TDZ (1.5 mg L−1), resulting in 5.3 ± 0.24 shoots of per explant and shoot length of 6.12 ± 0.19 cm. For the best in vitro root induction, the aseptic shoots excised from the culture flasks and inoculated individually on MS medium supplemented with 1.5 mg L−1 indole-3-butyric acid (IBA) resulted in 74.2% root growth and 8.82 ± 0.31 roots per explant. Although callus induction was observed in MS containing 2,4-dichlorophenoxyacetic acid (2, 4-D) and 1-naphthaleneacetic acid (NAA), the highest callus proliferation was observed with MS and NAA (2.0 mg L−1). Micropropagated plants established on soil showed 83% survival on acclimatization to the field conditions.

Efficient in vitro shoot bud proliferation from cotyledonary nodes and apical buds of Moringa oleifera Lam.

Moringa oleifera Lam. is a quick-growing tree species that has advantages for large-scale production including high yields, high nutrient contents, and diverse industrial applications. To assist such use, we have developed an efficient protocol for in vitro shoot bud proliferation from the plant’s cotyledonary nodes and apical buds. We also investigated the effects of the type of explants, various combinations of plant growth regulators and sucrose contents, and illumination intensity on the proliferation of both types of explants. The results showed that the most suitable tested medium for regenerating shoot buds from cotyledonary nodes was Murashige and Skoog (MS) basic medium supplemented with 1.0 mg/L 6-benzyladenine (BA) and 0.05 mg/L kinetin (KT). This yielded shoots from 66.7% of CNs, and 4.40 shoots per explant on average. In contrast, MS basic medium supplemented with 1.2 mg/L BA and 0.05 mg/L KT was the most suitable tested medium for regenerating shoot buds from apical buds, yielding shoots from 100% of the explants, with 4.13 per explant on average. In addition, increasing the sucrose content of the medium and illumination intensity inhibited their proliferation. MS medium with 0.02 mg/L α-naphthalene acetic acid was suitable for plantlet rooting. The efficient protocol based on these findings reported here may greatly facilitate industrial-scale in vitro generation of uniform seedlings and genetic improvement of the species.

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).

EFFECT OF SEASON AND GROWTH REGULATORS ON IN VITRO PROPAGATION OF JACKFRUIT (ARTOCARPUS HETEROPHYLLUS LAM)

The present study was conducted at the Laboratory of Plant Tissue Culture of Agrotechnology Discipline, Khulna University, Khulna, during the period from September, 2004 to May, 2005 with a view to study the effect of time of the year and growth regulators on in vitro propagation of jackfruit. Shoot tips from fresh sprouts on the trunk of mature jackfruit trees (Artocarpus heterophyllus Lam.) were used as explants. The explants were collected at three different times of the year from the same jackfruit plants viz. 5/09/04, 18/10/04, 5/01/05. The collected explants were cultured on MS medium supplemented with NAA-0.5 mgl-1 and fortified with different levels (1.0, 1.5 and 2.0 mgl-1) of BA to study the proliferation and multiplication of shoots from the cultured shoot tips and their subsequent growth and development. The in vitro shoot bud proliferation, multiplication and survivability were found better when the explants were cultured in January. BA at a concentration of 1.5 mgl-1 was found most suitable for shoot proliferation and multiplication. And all the three concentrations of BA varied significantly. The survivability of the proliferated shoot buds was found better in concentration of BA at 1.0 and 1.5 mgl-1.

Melatonin-induced Rhizome Proliferation, Differentiation, and Rooting during Rapid Propagation of Cymbidium goeringii and Cymbidium faberi

Melatonin plays an important role in plant resistance to stress. The role of melatonin in the propagation of plant tissue, such as rhizome proliferation, differentiation, and seedling rooting in Cymbidium species, remains unknown. In this study, we selected C. goeringii and C. faberi as experimental materials and attempted to understand the effect of melatonin on this process. We found that 1.0 μM melatonin was beneficial for rhizome proliferation of C. goeringii, with a proliferation rate of 5.52. In terms of C. faberi, the highest proliferation rate of 8.29 was observed in the medium with 0.5 μM melatonin. In proliferation, the cut rhizome of C. goeringii is more likely to cause browning phenomenon than that of C. faberi. The addition of melatonin can significantly inhibit the browning phenomenon and improve the survival rate of C. goeringii rhizome. The highest number of shoot buds per explant (3.11 after 60 days) was observed in the medium with 1.0 μM melatonin. The number of shoot buds per explant (3.28 after 60 days) was significantly higher for C. faberi in the medium with 5.0 μM melatonin than that for the control. Furthermore, culture medium incorporated with 1.0 μM of melatonin had the best comprehensive effect of seedling height and root number and length of C. goeringii. By contrast, 0.5 μM melatonin significantly promoted root elongation of C. faberi, reaching 1.77 cm, whereas it was 0.28 cm in the control. We demonstrated that melatonin in specific concentrations effectively promote rhizome proliferation, differentiation, and seedlings rooting in the rapid propagation of C. goeringii and C. faberi.

Simple and robust multiple shoot regeneration and root induction cycle from different explants of Hypericum perforatum L. genotypes

Hypericum perforatum L. commonly known as Saint John’s Wort (SJW) is an economically important medicinal plant known for accumulating its valuable bioactive compounds in a compartmentalized fashion. The dark glands are very rich in hypericin, and translucent glands are filled with hyperforin. The antibiotic properties of the afore mentioned bioactive compounds make it hard to establish tissue regeneration protocols essential to put in place a transformation platform that is required for testing gene function in this challenging species. In this study, we report the establishment of a regeneration and root induction cycle from different types of explants. The regeneration cycle was set up for the continuous supply of roots and leaf explants for downstream transformation experiments. The most effective medium to obtain multiple shoot-buds from node cultures was MS (Murashige and Skoog, Physiol Plant 15:473–497, 1962) medium supplemented with 0.5 mg L−1 6-Benzylaminopurine (BAP) and 0.5 mg L−1 indole-3-butyric acid (IBA). The same combination yielded copious amounts of shoots from root and leaf explants as well. For rooting the elongated shoots, MS medium devoid of plant growth regulators (PGRs) was sufficient. Nevertheless, addition of a low amount of IBA improved the quantity and quality of roots induced. Additionally, the roots obtained on a medium containing IBA readily developed shoot buds.

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.

The Dynamics of Non-Structural Carbohydrates in Different Types of Bamboo in Response to Their Phenological Variations: Implications for Managing Bamboo Plantations

Non-structural carbohydrate (NSC) dynamics in different bamboo types underlie the different management practices of bamboo plantations. The changes in moisture, soluble sugars, and starch content in response to phenological variations in different types of bamboo were measured to reveal the NSC dynamics in different bamboo forests for high yield. It was reported for the first time that new leaves of monopodial bamboo started sprouting after the completion of branching, while sympodial bamboo extended new leaves and branches simultaneously. NSC accumulation for shooting lasted about eight months for monopodial bamboo but only a short interval of two to three months for sympodial bamboo. The moisture content in different types of bamboo showed a similar trend of increasing from March to July and then decreasing until the coming January. From March to May, particularly in March, irrigation was required for the shooting of monopodial bamboo, whereas the shoot buds in sympodial bamboo showed sprouting, branching, and leafing. Total NSCs in different bamboo types remained relatively high in March, gradually decreased to the lowest in July, and then increased continuously until the coming January. Average soluble sugar and starch contents showed the same trend as the total NSCs in different types of bamboo. During the period from March to July, large amount of photoassimilates were required for shooting and height growth and then for branching and leafing in monopodial bamboo. Abundant photoassimilates were also required for branching, leafing, and shoot bud differentiation, as well as for subsequent shoot germination in sympodial bamboo. Thus, fertilizer application to both monopodial and sympodial bamboo plantations should be scheduled in early May and late July. Given the moisture, soluble sugar, starch, and total NSC content, January was the best season for harvesting monopodial bamboo for high production and future growth, whereas for sympodial bamboo, November was the best season.