Axillary Bud Proliferation Research Articles

Micropropagation and in vitro rejuvenation of Eucalyptus cloeziana F. Muell.

Micropropagation is an important tool for the propagation for recalcitrant species, like Eucalyptus cloeziana. This is a first reported about a complete study of the E. cloeziana, it includes the vegetative rescue protocol of mature trees, its micropropagation up to the implementation of a mini-garden and clonal micro-garden and evaluation of adventitious rooting of their propagules. The in vitro rejuvenation of E. cloeziana genotypes (03, 05, 06, 13 and 15), over 21 successive subcultures, was evaluated by comparing minicutting and microcutting techniques and the efficiency of this biotechnological tool for cloning of the mature trees. The results provide evidence of the in vitro rejuvenation occurring in the mature E. cloeziana trees during micropropagation, leading to increases in the multiplication ratios of the microstumps of three genotypes (03, 05 and 15). The survival and callogenesis percentages of the mini and microcuttings of E. cloeziana were higher than 80% upon exiting of the greenhouse (30-days old) in the three cutting times. The results suggesting that factors beyond tissue maturity influence the adventitious rooting of the E. cloeziana because minicuttings and microcuttings of the 05 and 13 elite genotypes exhibited rooting percentages lower than 20%. These findings provided a theoretical basis for realizing the micropropagation of the epicormic shoots of the mature trees and, subsequently, its in vitro rejuvenation by axillary bud proliferation.

In vitro propagation of triploid Toxicodendron vernicifluum (Stokes) F. A. Barkl

AbstractTriploid lacquer trees have a higher yield of raw lacquer and greater stress resistance; however, it is challenging to develop efficient and rapid seedling culture techniques because of the influence of metabolites with raw lacquer. In this study, the stem segments of a triploid lacquer tree were used to investigate the effects of explant sterilization treatment, different combinations of exogenous hormone concentrations on the induction of axillary buds, seedling development, and rooting, as well as transplanting substrate ratios. The results showed that the optimal combination of disinfection treatment for triploid Toxicodendron vernicifluum (Stokes) F. A. Barkl explants involves 75% alcohol immersion for 10 s, followed by 2.0% benzalkonium chloride treatment for 1 min and 0.1% HgCl2 treatment for 4 min. The suitable medium for axillary bud induction was Murashige and Skoog (MS) containing 0.1 mg•L−1 zeatin (ZT) and 0.1 mg•L−1 6-benzylaminopurine (6-BA), resulting in a high induction rate of 95.0%. Use of 1/2 MS supplemented with 0.5 mg•L−1 6-BA and 0.3 mg•L−1 2,4-dichlorophenoxyacetic acid (2,4-D) was the most effective for axillary bud proliferation. The combination of 1/2 MS containing 0.1 mg•L−1 6-BA, 0.5 mg•L−1 2,4-D, 0.5 mg•L−1 indole-3-butyric acid (IBA), and 0.2% activated carbon (AC) was an optimum for hardening-off. With woody plant medium (WPM) supplemented with 0.5 mg•L−1 naphthaleneacetic acid (NAA) and 0.5 mg•L−1 2,4-D and 0.5 g•L−1 AC as a rooting medium, the rooting rate was 67.8%. Also, the appropriate transplanting substrate ratio for triploid Toxicodendron vernicifluum (Stokes) F. A. Barkl was grass charcoal:laterite:humus = 1:1:1. Collectively, the results provided the technical assurance for seedling scale multiplication and propagation, facilitating its application in production.

APPLICATION OF ETIOLATION OF GRAPE EXPLANTS IN VITRO CULTURE

Micropropagation of grapes is used to obtain original and certified planting material for planting mother plants. The decrease in the efficiency of microclonal reproduction is associated with difficulties at the stages of introducing explants into in vitro culture and rooting. The method of etiolation is simple to use and allows you to prevent phenolic oxidation of explants, has a positive effect on increasing the length of shoots and the number of roots. The purpose of the research was to determine the effectiveness of etiolation at the stages of introducing explants and rooting microcuttings in in vitro culture. The work was carried out in the laboratory of in vitro grape culture of the department of grape nursery, propagation and biotechnology of the National Scientific Centre «V.Ye. Tairov Institute of Viticulture and Winemaking» NAAS of Ukraine according to the generally accepted method on table, technical and grafted varieties of grapes. Etiolation was applied in two stages: introduction of initial grape explants into in vitro culture (for 7, 15, and 25 days) and rooting of grape microcuttings (for 7 and 15 days). The survival and proliferation of explants, the main biometric indicators of growth and development of microclones were determined. It was established that at the stage of introduction of initial explants of grapes into in vitro culture, the use of etiolation for 15 days, and at the stage of micropropagation - for 7 days, had a positive effect on rooting, proliferation of axillary buds and further development of explants and microcuttings. The height of the stem of the experimental microclones was 0.6-2.6 cm higher, and the number of leaves was 0.4-0.9 more than that of the control plants. Also, etiolation had a positive effect on the increase in the length and biomass of roots in microcuttings. In the future, it is planned to analyze changes in the in vitro reproduction coefficient of various grape varieties under the influence of etiolation.

Micropropagación de piña (Ananas comosus L. Merr) variedad MD2 mediante proliferación de yemas axilares

The pineapple (Ananas comosus L. Merr) is an economically important bromeliad plant due to the organoleptic characteristics of its fruit, being the MD2 variety one of the most cultivated. Pineapple culture is limited by its inefficient sucker production. Micropropagation represents a way of producing pineapple plantlets. The aim of this study was to obtain a micropropagation protocol for A. comosus MD2 variety using the axillary bud proliferation technique. Axillary buds from suckers were put into MS media where 17 treatments were tested: benzylaminopurine (BA) in combination with indolebutyric acid (IBA) (0, 0.5, 1 mg/L) or indoleacetic acid (IAA) (0, 0.5, 1 mg/L); as well as BA (2 mg/L) with naphthaleneacetic acid (NAA) (5 mg/L); and kinetin (KIN) (2 mg/L). The presence or absence of silver nanoparticles (34 mg/L) was tested as well. The percentage of sprouting, number of shoots, and length were measured. Percentage of sprouting was higher (73.3%) when kinetin and silver were present. Highest shoot number (2.5±0.49) was observed on media containing BA 3 mg/L and AIA 0.5 mg/L. Also, organogenesis was observed on media with BA and ANA. Finally, acclimatization had a 100% success rate.

Mango malformation: etiology, symptoms, distribution and cultivar susceptibility in the Dominican Republic

Mango malformation disease (MMD) caused by Fusarium species has become an important disease of mango ( Mangifera indica L.) orchards worldwide. In the Dominican Republic, MMD was first observed in 2008 in orchards of Azua and Peravia provinces, with incidence of up to 50%. This study aimed to estimate the malformation prevalence and distribution in the main mango growing areas of the Dominican Republic and to assess the susceptibility of six commercial cultivars commonly grown in this country. Mango malformation was found in 18 out of 58 orchards, all located in Peravia and Azua. Peravia showed the highest incidence, with values of up to 75% (2013) and 87.5% (2014), and the interpolative analysis made with ArcGIS showed this province as being more inductive for Fusarium spp. occurrence. Pathogenicity tests were conducted in 2013 and 2014 on 10-month-old healthy mango plants per cultivar. Inoculation was done by infiltration of Fusarium pseudocircinatum and Fusarium decemcellulare conidial suspensions into the vegetative apical buds, singly and in combination. In inoculated plants, symptoms such as loss of apical dominance, excessive proliferation of apical or axillary buds with thickened stems, and reduction of leaf lamina were associated with F. pseudocircinatum. In addition to these symptoms, leathery protuberant buds and eventual death of infiltrated plants were associated with F. decemcellulare, singly or coinoculated with F. pseudocircinatum. Based on a disease severity index (DSI), “Tommy Atkins” was tolerant (DSI = 7.8%) and “Puntica” and “Banilejo” were highly susceptible (DSI = 22% and 28.5%).

Large-Scale In Vitro Propagation and Ex Vitro Adaptation of the Endangered Medicinal Plant Eryngium maritimum L.

The endangered medicinal plant Eryngium maritimum L. faces significant natural and anthropogenic threats. Therefore, in vitro propagation is recommended for both conservation and commercial purposes. The aim of the study was to develop a series of protocols for seed disinfection, in vitro multiplication and rooting, and ex vitro and field adaptation. For explant disinfection, the length and temperature of three consecutive disinfectants were investigated. Macrosalt modifications of MS medium and plant growth regulator addition to media effect on axillary bud propagation rate and rooting was studied. Survival and leaf growth during ex vitro and field adaptation in response to potting media, pot cell diameter, and light spectrum were tested. Seeds treated with diluted detergent at 40 °C for 180 min, followed by 0.01% KMnO4 for 3 h and commercial bleach for 11 min, achieved a lower rate of contamination and high germination rate. Axillary bud proliferation and rooting were enhanced by reducing nitrogen content in media and adding plant growth regulators. Potting media and pot size affect survival and growth ex vitro. Timely transplantation to field conditions before overwintering increases plantlet survival. In the present work, a suitable foundation is laid to scaleup the production of E. maritimum by micropropagation.

MICROPROPAGATION OF PHARMACEUTICAL CROP RUBIA CORDIFOLIA

Plants are cherished as the chief resource for healthcare products attributed to synthesizing diverse array of bio-molecules through various metabolic alterations. Over exploitation of such resources can intimidate its existence. Developing medicinal plants into crops and the use of micropropagation as technique to mass produce high-demand biomass can make a continuous supply of natural therapeutic products. Rubia cordifolia is one such plant, so an improved, efficient, reproducible and reliable in vitro clonal propagation protocol is to be adopted for the conservation of germplasm. MS medium fortified with phytohormons viz., BA, Kin, 2-iP, TDZ, mT, IAA, IBA and NAA singly or in combinations were used for the study. After one month of inoculation, 2.5 μM of BA and TDZ (9.37) endorsed the instigation of multiple shoots through axillary bud proliferation in a short span of time. In vitro produced microshoots were subjected to ex vitro rooting by treating 2.5 mM IBA for 5 min and planting in plastic pots containing potting mixture, to produce maximum rooting (79.17%) response. It was found to be an efficient and effective mode of rooting, since it combines both hardening and rooting. Under green house condition, all the rooted plantlets showed more than 90% survival. So direct regeneration protocol from nodal explants without the intervention of callus and thus ensure production of clonally uniform plants of R. cordifolia.

Micropropagation of Seed-Derived Clonal Lines of the Endangered Agave marmorata Roezl and Their Compatibility with Endophytes.

Simple SummaryThe wild Agave marmorata Roezl has been classified as an endangered species. Extracting these plants from the forest for commercial purposes and long maturation periods of close to 30 years have contributed to their loss. A. marmorata interacts with pollinators and other Agaves species to maintain genetic variability. Thus, the conservation and restoration of the agave ecosystem is an ecological challenge. Typically, agave micropropagation use meristem or leaves as explants to rapidly produce uniform agave plants in age and size on a large scale leading to homogeneous plantations. However, introducing these clones to the field reduces genetic variability. This study evaluated in vitro micropropagation of A. marmorata from seeds to generate clonal lines. The selected seedlings exhibited variations in multiplication capacity and stable tissue formation. Variations in clonal lines could be exploited to produce high-quality plants with different capacities, such as faster propagation, enhanced stress adaptation, and continued growth under nutrient limitation conditions, consequently maintaining genetic variability. Furthermore, some clonal lines were inoculated with four endophytic bacteria to identify other differences among these plants, including endophyte-host compatibility. Variable responses to inoculation were observed among clonal lines. We found that Achromobacter xylosoxidans was compatible, unlike Enterobacter cloacae which caused plant death.A. marmorata is the raw material used for tepextate mescal production but is classified as an endangered species. In the present study, we obtain and multiply clonal lines of Agave marmorata Roezl by selecting seedlings derived from seeds. Ten seedlings from two lots of 400 germinated seeds were selected for axillary bud proliferation induced by BAP 5 mg/L in vitamin-free Murashige and Skoog’s medium. Differences in shoot numbers, heights and senescent tissue formation were observed. Notably, the AM32 line formed 84 shoots and presented low senescent tissue after 60 d of culture. We also selected the AM31 and AM33 clonal lines. Four-month shoots were extracted with 80% methanol in water to determine the total content of saponins, flavonoids, and phenolic acids and compare the three clonal lines. Some bioactive molecules were identified using HPLC techniques and MALDI-TOF mass spectrometry none showed significant differences in content. Additionally, plants derived from the clonal lines were inoculated with four endophytic bacteria. Among these, Achromobacter xylosoxidans supported plant growth of AM32. A notable effect of plant death was observed after inoculation with Enterobacter cloacae, an endophyte of A. tequilana. Additionally, Pseudomonas aeruginosa, an endophyte from A. marmorata, reduced biomass. Our results demonstrate the incompatibility of A. marmorata to E. cloacae and specialization between the host plant and its endophytes. The compatibility of the plant-endophyte could be exploited to boost the establishment and stability of mutualisms to benefit plant development, stress tolerance and pathogen resistance. The differences in multiplication capacity, stable tissue formation, and endophyte biotization responses may indicate genetic variability. Clonal selection and micropropagation from seed-derived plants could contribute to conserving the endangered A. marmorata plant for reforestation in their natural habitats, thus, assuring mass propagation for sustainable industrial production of mescal, bioactive compounds, and prebiotics.

Effects of Sucrose and Myo-inositol on In Vitro Shoot Multiplicationof Promising Interspecific F1 Hybrid of Eucalyptus (Eucalyptus Tereticornis X Eucalyptus Grandis)

Abstract –Micropropagation technique is standardized for its multiplication, using nodal segments of 25-30 years old trees i.e. promising interspecific F1 hybrid of Eucalyptus (Eucalyptus tereticornis X Eucalyptus grandis). 0.1% Mercuric chloride solution for 10-15 minutes used for surface sterilization of nodal segments followed by 0.1% fungicide treatment for 1 minute and then washed 4-5 times with sterilized distilled water. These surface sterilized nodal segments were cultured on MS medium combination with auxin and cytokinin (NAA + BAP) for axillary bud proliferation. MS medium with combin- ation of 1.5mg/l BAP + 0.1mg/l NAA gave optimum rate of axillary bud induction. The in vitro shoot were cultured on MS medium with different concentration of BAP (0.1–3.0 mg/l) alone or in combination with NAA (0.1-1.5mg/l) and supplemented with sucrose at 3% level was the best for the growth and development of shoots. These proliferated axillary shoots were excised and subcultured on MS + 1.0 mg/l BAP + 0.1mg/l NAA medium to proliferate in vitro shoots.

Buds Reactivity and Factors Promoting Shoots Proliferation and Rooting of Cashew Seedlings Using <i>in Vitro</i> Tissue Culture Process

Tissue culture techniques are widely used for the mass propagation of many species. In cashew in vitro propagation, some protocols need to be established at this end. The present work was carried out to evaluate the conditions for in vitro regeneration of cashew seedlings from micropropagation by organogenesis on Benin genotypes. Nodal explants from one-month-old cashew seedlings in the greenhouse and cotyledonary nodes from in vitro germination were used for this purpose. BAP and kinetin were evaluated alone at 2.2 mg/L and then the combination of 2.2 mg/L BAP + 0.2 mg/L IBA was also evaluated. The response of axillary bud proliferation on explants was obtained with both cotyledonary nodes and axillary buds from different combinations of growth regulators. However, the best responses were recorded with cotyledonary nodes. When 2.2 mg/L BAP was used, 80% of the explants responded with numerous proliferation (5 to 8) buds (5.75 ± 0.12) with good shoot length (6.73 ± 0.3 cm) on MS medium containing 150 mL coconut water. Rooting was observed with the combination of NAA (2.5 mg/l) + IBA (2.5 mg/l) on 1/2 MS containing 40 g/l sucrose.

In vitro Propagation and Caulogenesis of Dracaena draco Plants

Dracaena draco is a monocot from family Asparagaceae. The dragon tree is a subtropical plant and features many botanic and Mediterranean gardens worldwide. D. draco has been categorized as vulnerable in Europe and endangered worldwide in the IUCN Red List of Threatened Species (IUCN, 1998). The aim of this study is to develop an efficient protocol for rapid in vitro propagation and caulogensis of D. draco Plants via enhanced axillary bud proliferation from single nodal explants cultured on full strength MS medium, 30g/l sucrose, 4g/l gelrite augmented with various concentrations of plant growth regulators as BA, NAA, IBA and their combinations. In general, the present study concluded that the best results for the initiation stage were recorded on MS medium fortified with NAA and BA at 2.00 and 1.00 mg/l, respectively. Meanwhile, supplemented medium with BA and NAA at 4.00 and 1.00 mg/l, respectively, gave rise to the best results for the multiplication stage. Regarding rhizogenesis stage, the best results were recorded when the neoformed shoots of the multiplication stage were divided singly and cultured on MS medium plus IBA and NAA at 1.00 and 0.50 mg/l, respectively which led to the highest mean number of roots formed per propagule. For callus induction, the internodal segments cultured into MS medium supplemented with BA and NAA at 0.50 and 2.00 mg/l, respectively, gave rise to the best results for the percentage of explants formed callus and callus size. Concerning the percentage of direct organogenesis and number of shoots formed per explants best results obtained from BA and NAA at 0.250 mg/l and 0.00 mg/l, respectively.Neoformed plantlets were acclimatized ex vitro and in vivo vigorously in a mixture of perlite and peat moss at (1:1) or (2: 2) or (2:3) and (3:3) consecutively, in addition to fixed volume (1 portion) of sterile sand; which resulted in the highest mean value of survival percentage/plant (100%) and showed true-to-type plants ex vitro.

Correction to: Synergistic effect of cytokinins and auxins enables mass clonal multiplication of drumstick tree (Moringa oleifera Lam.): a wonder

The synergistic effect of plant growth regulators on axillary bud proliferation for mass clonal multiplication of Moringa oleifera Lam. (vern. drumstick) has been assessed for the first time. Treatment of decoated seeds with 1% (w/v) Bavistin for 60 min, 0.33% (w/v) streptocycline for 30 min, and 0.1% (w/v) HgCl2 for 3.5 min resulted in complete removal of the surface contaminants. Maximum seed germination (89.13%) was obtained on quarter-strength Murashige & Skoog (MS) medium. Culture of nodal segments on MS + 6-benzyladenine (BA) at 3 mg L−1 resulted in multiple shoot proliferation with ~ 18 shoots per explant. All combinations of indole-3-acetic acid (IAA) + kinetin (Kn) resulted in elongated shoots, while only lower concentrations of BA (0.5 mg L−1), along with IAA (0.5 to 2 mg L−1), or Kn (0.5 to 5 mg L−1), showed significant synergy in the shoot morphogenesis. In addition, the maximum (100%) rooting efficiency was attained on half-strength MS medium supplemented with different concentrations of IAA and indole-3-butyric acid (IBA). The rooted plants were successfully established in the greenhouse for acclimatization. Clonality of the raised plants was assessed using 15 random primers of Operon® technologies (OPT and OPF series), and eight primers resulted in significant amplification with distinct, identical, and reproducible bands that confirmed clonality of the micropropagated plants. The present study provides a comprehensive analysis of the synergistic effect of plant growth regulators (PGRs) on in vitro shoot regeneration and proliferation for clonal mass multiplication disease-free plantlets, which can be utilized to maximize the yield of healthy and genetically identical plants of drumstick tree, which is considered to be a miracle multipurpose tree.

Alteration of media enables efficient in vitro cloning of mature Elaeocarpus serratus L. (Ceylon olive): a commercially important fruit tree.

Elaeocarpus serratus is a fruit tree able to propagate through conventional vegetative means to a limited extent restricts its wide cultivation by the farmers. In the present report, we have developed an efficient in vitro propagation protocol using mature nodal explants from a 17-year-old tree for the first time with 6.6 shoots/culture. Explants cultured on agar (0.8%) gelled standard Murashige and Skoog (MS) medium, ½ MS, ¾ MS, White's, Gamborg's B5 or woody plant medium (WPM) supplemented with 2.5µM benzyl adenine (BA) and 0.1µM α-naphthalene acetic acid (NAA) showed the superiority of ½ MS medium in terms of explant response and number shoots (6.6). Further optimization of ½ MS medium by altering nutrient elements (macros, micros, vitamins and Fe EDTA) were undertaken, and MS medium composed of half-strength major salts, original strength of minor salts and vitamins were supplemented with BA (2.5µM) and NAA (0.1µM), produced enhanced axillary bud proliferation (8.88/explant) and shoot elongation (3.83cm). Reculturing of original explant on this medium after IV passages produced more than 16 healthy shoots per culture which attained a length of 4.13cm. Microshoots raised through this way were rooted (86.11%) ex vitro by pulse treatment with 2mM indole-3-butyric acid (IBA) for 5min followed by planting in nursery pots containing a 1:1:1 (v/v/v) mix of sand, soil, and farmyard manure. The hardened plants were successfully planted in the fruit tree garden of the Department. Genetic fidelity of micropropagated and mother plants were tested using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers which showed a high degree of monomorphism thus supported morphological uniformity of micropropagated plants.

Molecular identification of a ‘Candidatus Phytoplasma australasia’ strainin radish showing a little leaf disease in Andhra Pradesh, India

Symptoms of little leaf, proliferation of axillary buds and pale green colour leaves were observed in radish plants grown in the farmer fields at Anantapuram, Andhra Pradesh. DNA was extracted from symptomatic and asymptomatic leaves and subjected to nested PCR assay with P1/P7 and R16F2n/R16R2 primers. The about 1,250 bp amplicon was obtained only from symptomatic radish plants and the positive control, but not from any asymptomatic plants. Pair wise sequence comparison and phylogenetic analyses of 16S rRNA gene sequences of the radish phytoplasma strain suggested that it is related to ‘Candidatus Phytoplasma australasia’. Further virtual RFLP analysis of 16S rDNA sequences allowed the classification of the radish phytoplasma strain in the 16SrII-D subgroup that results to be in great expansion in this area of India.

Genetic fidelity assessment of long term in vitro shoot cultures and regenerated plants in Japanese plum cvs Santa Rosa and Frontier through RAPD, ISSR and SCoT markers

In vitro propagation can be explored to overcome the constraint of limited planting material in plum. Although it ensures the production of plants in large number, but there are chances of somaclonal variations using this technology. Thus, clonal fidelity of in vitro raised plants should be checked to obtain true to type planting material prior to transplantation in the field. Genetic fidelity of in vitro cultures of plum (Prunus salicina) cvs. Santa Rosa and Frontier multiplied for 5 years (60 passages) through enhanced axillary bud proliferation was tested and compared with 2 year old in vitro raised and mother plants of respective cultivars using RAPD, ISSR and SCoT markers. Out of twenty-eight RAPD primers, eighteen produced 29 and 27 distinct bands in Santa Rosa and Frontier whereas, all of the fifteen ISSR primers screened generated clear reproducible bands in both the cultivars. In SCoT assay, eight primers out of twenty-six generated reproducible bands in both the cultivars. Homogenous amplification was observed in all the samples thereby confirming the genetic fidelity of tissue culture raised plants, thus suggesting that in vitro propagation using axillary buds is the safest mode for the production of clonal planting material in plum.

IMPROVEMENT OF THE PROCESSES OF MICROCLONAL REPRODUCTION OF BLACKBERRY RUBUS CAESIUS L. VAR. THORNFREE

Aim. The initial stages’ improvement of microclonal propagation of Blackberry Rubus caesius L. var. Thornfree, particularly the introduction to in vitro culture for subsequent effective cultivation. Materials and methods. Methods of introduction of initial explants of blackberry to in vitro culture and methods of microclonal reproduction were used. Two schemes of sterilization of plant material of Blackberry with the use of fungicidal drugs Hinozol and Horus were tested to identify the most optimal for this type of plants. The effect of agar concentration in the medium, the consistency of the nutrient medium on the processes of survivability, bud proliferation and induction of multiple shoots of Blackberry was studied, and semi-liquid medium was used for the first time. The medium was prepared semi-liquid (4 g/l of agar) and solid (8 g/l of agar). Results. The optimal sterilization scheme of plant material for the introduction of Blackberry to in vitro culture was determined and the most effective fungicidal drug – Horus was selected. It was found that the use of semi-liquid Murashige and Skoog nutrient medium for the initial stages of microclonal propagation of Blackberry to in vitro culture allows: to increase the survival of explants by 30%compared to solid medium; to accelerate the proliferation of axillary buds for 1–2 days; to increase the intensity of the additional shoots formation of Blackberry in vitro in 6–7 times. Conclusion. During the initial stages of microclonal propagation of Blackberry Rubus caesius L. var. Thornfree, it is advisable to use semi-liquid nutrient medium to improve the survival of explants, faster proliferation of buds and the formation of new multiple shoots in vitro.

In Vitro Propagation of an Endangered Helianthus Verticillatus by Axillary Bud Proliferation.

Helianthus verticillatus (Asteraceae), whorled sunflower, is a perennial species restricted to a few locations in the Southeastern United States. Habitat loss has caused H. verticillatus to become rare, and since 2014, it has been federally listed as an endangered species. As a part of the recovery plan for the restoration and protection of H. verticillatus, an efficient micropropagation protocol based on axillary shoot proliferation was developed. Various concentrations of 6-benzylaminopurine (BAP; 0 to 4.44 µM) were examined for their morphogenetic potential in the regeneration of six genotypes of H. verticillatus from the nodal explants derived from greenhouse-grown plants. Both the BAP concentration and genotype had significant effects on the regeneration capacity of H. verticillatus. Although the induced buds were observed on ½-strength Murashige and Skoog medium without plant growth regulators, a higher rate of induction and bud development were achieved on media with either 0.88 or 2.22 µM BAP, regardless of the genotype. Successful rooting of the induced shoots was achieved within four weeks after the transfer from the induction medium to the fresh ½-strength MS medium, but the rooting efficiency was dependent on the plant’s genetic background. Regenerated plantlets, with well-developed shoots and roots, were acclimatized successfully to greenhouse conditions with a 97% survival rate. Simple sequence repeats (SSRs) markers were employed to assess the genetic uniformity of the micropropagated plants of H. verticillatus. No extraneous bands were detected between regenerants and their respective donor plants, confirming the genetic fidelity and stability of regenerated plants. To our knowledge, the protocol developed in this study is the first such report for this endangered species.

Micropropagation, encapsulation, and conservation of Decalepis salicifolia, a vanillin isomer containing medicinal and aromatic plant

An efficient in vitro propagation and synthetic seed production protocol was established for the conservation of Decalepis salicifolia (Bedd. ex Hook.f.) Venter, an endemic and critically endangered ethnomedicinal species. Murashige and Skoog (MS) media supplemented with 2.2 μM BAP and 5.7 μM IAA produced shoot tip proliferation with an average shoot length of 5.81 ± 0.03 cm in 3 wk. Axillary bud proliferation was found to be best in 11.1 μM BAP with an average of 3.5 ± 0.06 shoots per explant. Shoot tip and nodal explants were encapsulated in sodium alginate and their regeneration was achieved following storage at 4°C up to 12 wk. Successful rooting was obtained in modified MS medium with low nitrate and high sucrose concentration. Quarter strength MS media containing 2.5 mM each of NH4NO3 and KNO3 along with 8% sucrose produced an average number of 12.4 ± 1.18 roots with an average length of 4.3 ± 0.08 cm. The in vitro derived rooted plants were successfully hardened (92.8%) and established in field with 100.0% survival rate. Inter-simple sequence repeat (ISSR) markers proved the genetic fidelity among the in vitro grown plant showing 99.5% similarity with the mother plant. Micropropagation derived acclimatized plants produced 2-hydroxy-4-methoxybenzaldehyde in amounts similar to seed-derived field-grown plants of the same age. The in vitro propagation protocol developed for D. salicifolia can be utilized to reduce exploitation pressure from their natural habitats and augment ecorestoration, conservation, and cultivation of this critically endangered and industrially valuable plant. The synthetic seeds technique will serve as propagules in ex situ gene banks, as well as supports cost-effective germplasm conservation.

Synergistic effect of cytokinins and auxins enables mass clonal multiplication of drumstick tree (Moringa oleifera Lam.): a wonder

The synergistic effect of plant growth regulators on axillary bud proliferation for mass clonal multiplication of Moringa oleifera Lam. (vern. drumstick) has been assessed for the first time. Treatment of decoated seeds with 1% (w/v) Bavistin for 60 min, 0.33% (w/v) streptocycline for 30 min, and 0.1% (w/v) HgCl2 for 3.5 min resulted in complete removal of the surface contaminants. Maximum seed germination (89.13%) was obtained on quarter-strength Murashige & Skoog (MS) medium. Culture of nodal segments on MS + 6-benzyladenine (BA) at 3 mg L−1 resulted in multiple shoot proliferation with ~ 18 shoots per explant. All combinations of indole-3-acetic acid (IAA) + kinetin (Kn) resulted in elongated shoots, while only lower concentrations of BA (0.5 mg L−1), along with IAA (0.5 to 2 mg L−1), or Kn (0.5 to 5 mg L−1), showed significant synergy in the shoot morphogenesis. In addition, the maximum (100%) rooting efficiency was attained on half-strength MS medium supplemented with different concentrations of IAA and indole-3-butyric acid (IBA). The rooted plants were successfully established in the greenhouse for acclimatization. Clonality of the raised plants was assessed using 15 random primers of Operon® technologies (OPT and OPF series), and eight primers resulted in significant amplification with distinct, identical, and reproducible bands that confirmed clonality of the micropropagated plants. The present study provides a comprehensive analysis of the synergistic effect of plant growth regulators (PGRs) on in vitro shoot regeneration and proliferation for clonal mass multiplication disease-free plantlets, which can be utilized to maximize the yield of healthy and genetically identical plants of drumstick tree, which is considered to be a miracle multipurpose tree.

Transcriptome Analysis Reveals Key Pathways and Hormone Activities Involved in Early Microtuber Formation of Dioscorea opposita.

Chinese yam (Dioscorea opposita) is an important tuberous crop used for both food and medicine. Despite a long history of cultivation, the understanding of D. opposita genetics and molecular biology remains scant, which has limited its genetic improvement. This work presents a de novo transcriptome sequencing analysis of microtuber formation in D. opposita. We assembled cDNA libraries from different stages during the process of microtuber formation, designated as initial explants (EXP), axillary bud proliferation after three weeks (BUD), and microtuber visible after four weeks (MTV). More differentially expressed genes (DEGs) and pathways were identified between BUD vs. EXP than in MTV vs. BUD, indicating that proliferation of the axillary bud is the key stage of microtuber induction. Gene classification and pathway enrichment analysis showed that microtuber formation is tightly coordinated with primary metabolism, such as amino acid biosynthesis, ribosomal component biosynthesis, and starch and sucrose metabolism. The formation of the microtuber is regulated by a variety of plant hormones, including ABA. Combined with analysis of physiological data, we suggest that ABA positively regulates tuberization in D. opposita. This study will serve as an empirical foundation for future molecular studies and for the propagation of D. opposita germplasm in field crops.