Nodal Stem Segments Research Articles

Effect of Different Concentrations and Combinations of Benzyl Aminopurine and Indole-3-Butyric Acid on Micropropagation of <i>Vanilla Planifolia</i>

Micropropagation of explants in vitro has potency to address propagule demands for promoting large-scale vanilla production. Plant growth regulators (i.e., cytokinin, auxin) are necessary for the plant micropropagation success. Objective of this study is to determine the shoot multiplication and root development of Vanilla planifolia under the influence of different concentrations and combinations of BA and IBA for micropropagation. Sixty stem nodal segments of Vanilla planifolia were cultured on MS medium supplemented with IBA (0, 0.5, or 2.0 mg/L) and combined with BA (0, 0.5, 1.0, or 2.0 mg/L). Shoot multiplication and root induction were measured after 60 days of culture. The results show that the MS medium with 1 mg/L IBA hindered shoot growth, while the medium containing 1 mg/L BA yielded the highest number or weight of shoots per explant. For the root development, supplementing the medium with 0.5 mg/L or 1 mg/L IBA improved root length or number of roots per explant, respectively. This research establishes a valuable approach for vanilla micropropagation by utilising low concentrations of plant growth regulators and a rapid protocol. This paves the way for significant advancements in large-scale commercial production.

An Efficient and Rapid Protocol for Somatic Shoot Organogenesis from Juvenile Hypocotyl-Derived Callus of Castor Bean cv. Zanzibar Green.

Aseptic seedlings of different ages derived from surface-sterilized mature seeds were applied as an explant source. Various explants such as 7- and 21-day-old hypocotyl fragments, 42-day-old nodal stem segments, and transverse nodal segments of stem, as well as leaf petioles, were cultured on the agar-solidified Murashige and Skoog (MS) basal medium supplemented with 0.1 mg/L IAA, 5 mg/L AgNO3 and different types and concentrations of cytokinin (1 mg/L zeatin, 0.25 mg/L thidiazuron (TDZ), and 5 mg/L 6-benzylaminopurine (6-BAP)). Consequently, it was found that 7- and 21-day-old hypocotyl fragments, as well as nodal stem segments obtained from adult aseptic seedlings, are characterized by a high explant viability and callus formation capacity with a frequency of 79.7-100%. However, the success of in vitro somatic shoot organogenesis was significantly determined not only by the culture medium composition and explant type but also depending on its age, as well as on the size and explant preparation in cases of hypocotyl and age-matched nodal stem fragments, respectively. Multiple somatic shoot organogenesis (5.7 regenerants per explant) with a frequency of 67.5% was achieved during 3 subcultures of juvenile hypocotyl-derived callus tissue on MS culture medium containing 0.25 mg/L TDZ as cytokinin source. Castor bean regenerants were excised from the callus and successfully rooted on ½ MS basal medium without exogenous auxin (81%). In vitro plantlets with well-developed roots were adapted to ex vitro conditions with a frequency of 90%.

Induction and characterization of induced tetraploid, mixoploid and control diploid teak seedlings (Tectona grandis L.f.) from shoot cultures treated with oryzalin*

Teak, known for its high-quality timber and economic value, holds significant potential as the major component of an agroforestry system to combat climate change and support sustainable development goals. The challenge of slow teak growth can be addressed by introducing fast-growing teak varieties amenable to vegetative propagation. Furthermore, the improvement of teak through somatic cell genetics, such as inducing polyploidy, can result in more vigorous, faster-growing, and drought-tolerant trees. While a report exists on a tetraploid teak accession with enhanced less water tolerance, information on the induction and characterization of tetraploid teak is lacking. In this study, we report the induction of polyploid teak by immersing 1 – 1.5 cm nodal stem segments and shoot tips obtained from teak shoot cultures in a modified MS liquid medium containing 5 − 30 µM oryzalin for one week, followed by subculture for 6 passages. The resulting shoots were acclimatized and grown in the nursery for 10 months. Based on flow cytometry for ploidy analysis of pre-selected plants having morphologies typical of polyploid followed by chromosome counting, we identified two tetraploid plants from one accession and 18 mixoploid teak plants from four accessions. Chromosome counting analysis confirmed that a diploid plant had a chromosome number of 2n = 2x = 36, tetraploid plant had a chromosome number of 2n = 4x = 72, and mixoploid plant exhibited both chromosome number of 2n = 2x = 36 and 2n = 4x = 72 in root tip cells. Morphological characterization revealed that tetraploid and mixoploid teak plants displayed typical polyploid characteristics, such as larger, thicker, and greener leaves, increased root diameter, increased cell size in root tip region, and larger stomata size but with reduced density. The development of tetraploid teak holds the potential to provide faster-growing and more drought-tolerant varieties, facilitating breeding efforts, including the development of triploid varieties.

Propagation of Grape by Nodal Stem cuttings

Experiments were conducted to test the effect of MS saltsconcentration, potassium dihydrogen phosphate and growth regulators onshoot proliferation and growth in grapevine (Vitis vinifera L.), usingexplants derived from in vitro grown plantlets. Murashige and Skoogoriginal medium was used as basal medium. Shoot proliferation occurredprincipally when using nodal stem segments as explants. Among MS-saltstrengths tested, full MS- strength (1X) was optimum for shootproliferation and elongation. Increasing the original phosphorusconcentration in the medium by the addition of 170 mg/L KH2 PO 4 had apositive, marked effect on shoot number and shoot length. The nodal stemsegment explants responded best to relatively low concentrations of NAAand kinetin, and a large number of shoots and good plantlets height wereobtained on the medium containing 0.1mg/L NAA and 1.0 mg/L kinetin.Root formation occurred on the medium containing 0.3 mg/L IBA.

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.

Optimized Protocol for Micropropagation of Cadaman and Garnem Peach Rootstocks إستحداث بروتوکول للإکثار الدقيق لأصلي الخوخ الکادامان و الجارنم

The present study aimed to develop a micropropagation protocol for Cadaman (P. persica x P. davidiana) and Garnem (P. persica x P. dulcis) peach rootstocks. Stem nodal segments with one axillary bud used as explants. MS medium supplemented with different concentrations of BA with or without IBA was studied to determine the best for multiplication stage, The results revealed that MS medium supplemented with 2.0 mg/l BA+0.1 mg/l IBA was optimal for shoot proliferation of Cadaman rootstock, whereas MS containing 2.0 mg/l BA+ 0.0 IBA was advisable to Garnem peach rootstock. Concerning the effect of auxin type and concentration on the rooting stage, IBA at 1.0 mg/l was more effective on rooting induction of Cadaman shoots, whereas in case of Garnem rootstock both IBA and NAA auxins had a similar effect on the rooting process at the concentration of 1.0 mg/l individually. After acclimatization, survival rate was 83% and 63.3% for Cadaman and Garmen rootstocks, respectively.

Laser Microdissection of Specific Stem-Base Tissue Types from Olive Microcuttings for Isolation of High-Quality RNA.

Simple SummaryOnly a small portion of the stem cells participate in the process of adventitious root formation and the cells/tissues types involved in this process is species-dependent. In olive, it is still unclear which type of cells acquire competence for rooting. Regardless, the entire stem nodal segment (containing a mixture of distinct cell types) continues to be used in studies related to the molecular mechanisms underlying this process. Laser microdissection (LM) technology has been applied to isolate specific tissue and cell types. However, it is difficult to find a standard LM protocol suitable for all plant species and cell types and, thus, LM procedures must be developed and optimized for each particular tissue. In this study, we aimed to evaluate the efficiency of a LM protocol in olive microcuttings stem-base samples. This work presents a simple, rapid and efficient LM procedure for harvesting specific tissue types used for further high-quality RNA isolation. This will encourage future cell type-specific transcriptomic studies, contributing at deciphering rooting-competent cells in olive stems and to better understand the molecular mechanisms underlying the process of adventitious root formation.Higher plants are composed of different tissue and cell types. Distinct cells host different biochemical and physiological processes which is reflected in differences in gene expression profiles, protein and metabolite levels. When omics are to be carried out, the information provided by a specific cell type can be diluted and/or masked when using a mixture of distinct cells. Thus, studies performed at the cell- and tissue-type level are gaining increasing interest. Laser microdissection (LM) technology has been used to isolate specific tissue and cell types. However, this technology faces some challenges depending on the plant species and tissue type under analysis. Here, we show for the first time a LM protocol that proved to be efficient for harvesting specific tissue types (phloem, cortex and epidermis) from olive stem nodal segments and obtaining RNA of high quality. This is important for future transcriptomic studies to identify rooting-competent cells. Here, nodal segments were flash-frozen in liquid nitrogen-cooled isopentane and cryosectioned. Albeit the lack of any fixatives used to preserve samples’ anatomy, cryosectioned sections showed tissues with high morphological integrity which was comparable with that obtained with the paraffin-embedding method. Cells from the phloem, cortex and epidermis could be easily distinguished and efficiently harvested by LM. Total RNA isolated from these tissues exhibited high quality with RNA Quality Numbers (determined by a Fragment Analyzer System) ranging between 8.1 and 9.9. This work presents a simple, rapid and efficient LM procedure for harvesting specific tissue types of olive stems and obtaining high-quality RNA.

In Vitro Micropropagation of Rose (Hybrid Rosa spp.) through Plant Tissue Culture Technique

Roses are the most important cut flowers in the world. Tissue culture of rose has been improved since last twenty years, and exploited for various purposes from basic anatomical and physiological research to micropropagation from auxiliary buds, shoot tips, leaf explants, etc. Single bud nodal stem segments were surface sterilized and then cultured on MS medium supplemented with 30 g/l sugar, 6 g/l agar and different concentrations of BAP(1, 2, 3, 4 and 5mg/l). Among them, the most suitable concentration for shoot initiation and multiplication was 3mg/l BAP. And it also found that the best for shoot elongation occurred in MS medium supplemented with BAP 3 mg/l and GA3 1mg/l. The proliferated microshoots were transferred to root inducing media of half strength MS medium supplemented with 15 g/l sugar, 5 g/l agar and NAA (0.5, 1, 1.5, 2 mg/l). The best rooting was observed at NAA 1 mg/l concentration. The least response in root formation was found at NAA 0.5 mg/l supplementation on half strength MS media.

Improved Micropropagation of Stevia (Stevia rebaudianaBertoni) Plant

There is a great interest on the strategical plan in Egypt for the cultivation of a new crop used as source of natural sweeteners Stevia rebaudiana (Spanti) and (China-1) var. Stem nodal segments containing axillary buds were used as an explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of flurprimidol (flur.), paclobutrazol (PBZ) and thidiazuron (TDZ). In Spanti var. maximum number of branches (6.52) and (6.4) were obtained in MS medium supplemented with 0.12 mg/l flur. and 0.2 mg/l TDZ, respectively with an average of 56.4 and 36.67 leaves per plantlet, having an average shoot length of 6.40 and 6.52 cm, respectively. The best in vitro root induction (100%) was achieved on MS medium with 0.16 mg/l flur. with an average of 10.00 roots per plantlet and root length of 4.82 cm (Spanti var.). Furthermore, in China-1 var. (MS) medium supplemented with 0.16mg/l flur. induced the best morphological characteristics. The rooted plantlets from explant planted on (MS) medium supplemented with 0.16 mg/l flur. were successfully established in soil and grown to maturity at the survival rate of 100% in the greenhouse in (Spanti) and (China-1) var. As a result of anatomy, all studied growth regulators significantly enhanced the anatomical characters of stevia varieties leaf and stem.flur.at 0.16 mg\l surpassed, for instance, midvein and lamina thickness, length and width of leaf vascular bundle as well as stem diameter, xylem and phloem thickness. Our data revealed that the numbers of protein bands in most of treatments are bigger than which in the control.

Induksi Poliploidi Tanaman Kangkung (Ipomoea aquatica Forssk.) Kultivar Salina In Vitro dengan Oryzalin

ABSTRACT Water spinach is a vegetable plant consumed by people all over the world. Its small morphology requires improvement to increase the size and productivity. An alternative way to do that is by increasing the ploidy of its chromosome. This paper describes in vitro induction of polyploidy of water spinach. Inoculum of in vitro seedling, in vitro shoots and nodal stem segments were immersed in MS solution containing oryzalin at concentrations of 0.00, 1.25, 2.50, 3.75 and 5.00 µM with immersion duration of 4, 8 and 24 hours. Oryzalin treatments reduced the growth variables of the in vitro shoots of all inocula tested, compared to control of no oryzalin treatment. On immersion of 24 hours and high concentration of 5.00 µM, many inoculum failed to grow to the third passage of subculture. The best growth of in vitro shoot to the third passages occurred in the treatment combination of oryzalin 1.25 µM-8 h immersion and 2.50 µM-4 h immersion for seedling inoculum, treatment combination of 3.75 µM-4 h immersion for shoot tip inoculum and 1.25 µM-4 h immersion for nodal stem segment, compared to other treatments. Analysis of flow cytometry on 41 leaf samples from oryzalin treatment derived shoots showed 14.63% tetraploid, 36.59% mixoploid and 48.78% diploid. The efficiency of tetraploid formation reached 60%, provided only by the treatment of oryzalin 1.25 µM-8 h on seedling segments inoculum. Tetraploid shoots need to be proliferated, acclimatized, grown into planting materials and planted for agronomical analysis to provide evidence whether or not tetraploid water spinach is viable for commercial cultivation.

IN VITRO PROPAGATION FOR PEACH ROOTSTOCK (NEMAGUARD)

A procedure for micropropagation ofNemaguard peach rootstock was developed using stem nodal segments as explants for in vitroestablishment. Hundred percent of explants survived with high proliferation of shoots, growth percentage to survival and average shoot length was obtained on Murashige and Skoog (MS) basal medium supplemented with a combination of 0.5 mg/L 6-benzyl amino purine (BAP) plus 0.2 mg/L indole acetic acid (IAA). The maximum number of proliferated shoots (7.0 shoots/explant) was obtained on MS medium supplemented with 3.0 mg/L BAP + 0.5 mg/L 2-isopentenyladene (2iP). Meanwhile, MS basal medium without growth regulators induced the highest shoot length compared with other treatments. However, shoot elongation decreased with the increase of BAP concentration. Ninety percent of the shoots rooted on MS medium supplemented with 3.0 mg/L indole butyric acid (IBA) to obtain complete plants with the maximum average root number/shoot (5.8). On the other hand, the maximum root length (8.5 and 7.2 cm) was obtained when IBA or naphthalene acetic acid (NAA) were supplemented to half strength MS media at 3.0 mg/L. High survival, over 90%, was obtained when the plantlets were transferred to greenhouse conditions. The Nemaguard peach rootstock cans successfully micropropagated beginning with stem nodal segment without significant damage to mother plant. The in vitroNemoguard peach rootstocks were planted in Saint Catherine, South Sinai, Egypt where chilling requirements for producing the rootstock seeds are available for commercial production. Hence, it is encouraged to grow Nemaguard peach rootstock under S. Catherine conditions for commercial seed production.

In vitro propagation and assessment of genetic stability of acclimated plantlets of Cornus alba L. using RAPD and ISSR markers.

Cornus alba L. (white dogwood) is an important ornamental shrub having a wide range of applications such as reforestation programs and soil retention systems. The vegetative propagation of dogwood by cuttings may be slow, difficult, and cultivar dependent; therefore, an improved micropropagation method was developed. Nodal stem segments of C. alba cultivars ‘Aurea’ and ‘Elegantissima’ were cultured on media enriched with six different sources of macronutrients. Media were supplemented with either N6-benzyladenine (BA) or thidiazuron (TDZ) in combination with 1-naphthaleneacetic acid (NAA). Regardless of the cultivar, the best shoot proliferation was observed on Lloyd and McCown medium (woody plant medium (WPM)) at pH 6.2, containing 1.0 mg L−1 BA, 0.1 mg L−1 NAA, and 20–30 g L−1 sucrose. Rooting of regenerated shoots was achieved by an in vitro method when different concentrations of NAA or indole-3-butyric acid (IBA) were tested. Microcuttings were rooted for 8 wk on medium enriched with 0.25 mg L−1 NAA and potted into P9 containers in the greenhouse. The final survival rate of the plants after 20 wk was 80% for ‘Aurea’ and 90% for ‘Elegantissima’. Genetic stability of the micropropagated plants was confirmed by using two DNA-based molecular marker techniques. A total of 30 random amplified polymorphic DNA (RAPD) and 20 inter-simple sequence repeat (ISSR) primers resulted in 197–199 and 184–187 distinct and reproducible band classes, respectively, in ‘Aurea’ and ‘Elegantissima’ plantlets. All of the RAPD and ISSR profiles were monomorphic and comparable with the mother plant.

English

Handroanthus heptaphyllus (Vell.) Mattos (Bignoniaceae), a tropical forest tree, is a source of wood suitable for the manufacture of fine furniture and chemical compounds with medicinal and insecticidal properties. Poor seed viability of this species limits the conventional propagation practice. In vitro germination of seeds was achieved in semi-solid Murashige and Skoog’s (MS) medium. A disinfection protocol was optimized for germination and growth of seedlings in vitro . Proliferation of shoots from cotyledonary and stem nodal segments was achieved in semi-solid MS medium supplemented with 22.2 μM 6-benzyladenine. Temporary immersion culture promoted elongation and rooting of shoots and reduced the shoot tip necrosis compared with culture on semisolid medium (6.7±2.7 and 41.7±7.0%, respectively; P = 0.001). Plantlets derived from temporary immersion showed better performance during the acclimatization phase. This is the first report on micropropagation of H. heptaphyllus . Keywords: Seeds, uncoated seeds, cotyledon, stem nodes, axillary shoots, adventitious shoot, black lapacho

In vitro Propagation of Solanecio biafrae and Determination of Genetic Stability of Plantlets Using RAPD and ISSR Markers

Abstract An efficient and reproducible micropropagation protocol of Solanecio biafrae (Oliv. & Hiern) C. Jeffrey has been developed from nodal stem segments. Shoot development was obtained on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP) alone and in combination with zeatin and 1-naphthaleneacetic acid (NAA). Elongated shoots were rooted in the presence of zeatin or 3-indole-butyric acid (IBA) alone or in combinations. The highest number of explants forming shoots (100%) as well as the highest number of shoots per explant (3.4) and the longest shoots (22 mm) were recorded on medium containing 4.0 mg·dm−3 BAP, 2.0 mg·dm−3 NAA, and 1.0 mg·dm−3 zeatin. About 76% of shoots formed roots on half-strength MS medium free of plant growth regulators. The best root formation (approximately 88%) was recorded on the medium containing 1.0-1.5 mg·dm−3 IBA. The micropropagated shoots with well-developed roots were efficiently acclimatized under greenhouse conditions. The random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) amplification products were monomorphic in micropropagated plants and similar to those of mother plant showing their genetic uniformity. This is the first report of micropropagation of S. biafrae, which will facilitate in vitro mass propagation, conservation, and germplasm exchange of this endangered African vegetable.

Molecular Analysis of Drought Tolerance in Guava Based on In Vitro PEG Evaluation

Forty landraces of guava were subjected to drought tolerance evaluation based on in vitro polyethylene glycol (PEG) treatment. Nodal stem segments with lateral buds were used as explant. Five concentrations (i.e. 0, 4, 6, 8 and 10 %) of PEG were tested, among which the optimum concentration for screening was determined as 8 %. Analysis of variance showed highly significant variation among the tested landraces, concentrations of PEG and the interaction between them in the percentage of response (%R), number of shoot per explant (NSE) and drought susceptibility index. The average percentage of reduction due to PEG treatment was 48.30 and 52.57 % for the %R and NSE, respectively. Heritability and genetic advance were increased due to drought stress for %R, while they were decreased for NSE, indicating that %R was more related to drought stress than NSE. The molecular analysis of the highest and lowest responsive landraces was performed using sequence related amplified polymorphism (SRAP) and inter-simple sequence repeats (ISSR). Both markers were effective in discriminating the tested landraces and completely separated them into two clusters related to %R under PEG. ISSR showed a higher percentage of polymorphism, polymorphic information content and diversity index compared with SRAP. However, SRAP was more effective than ISSR in showing a higher primer resolving power and a number of unique specific bands for drought tolerance and susceptibility. Drought in vitro evaluation method established here is effective, inexpensive and manageable in genotype screening for drought tolerance in guava and could be used in other woody plant species.

In vitro Propagation of Crassocephalum crepidioides—An Endangered African Traditional Leaf Vegetable and Molecular Analysis of Micropropagated Plants

ABSTRACTCrassocephalum crepidioides (Benth) S. Moore is an important African traditional leaf vegetable, but its production is very low and its existence threatened. An efficient and reproducible micropropagation protocol of C. crepidioides was developed from nodal stem segments of pot-grown plants. Shoot induction was obtained on Murashige and Skoog (MS) medium supplemented with benzineamino purine (BAP) alone and in combination with zeatin and naphthalene acetic acid (NAA). Elongated shoots successfully rooted on zeatin alone and in combination with indole butyric acid (IBA). Molecular stability of micropropagated plants was evaluated using random amplified polymorphic DNA (RAPD) markers. Survival of nodal segments in micropropagation medium was greater than 70% in all treatments. The presence of plant growth regulators (PGRs) in MS medium influenced shoot formation, rooting, and number of shoots and roots per explant. The best shoot formation frequency (88.7%), number of shoots per explant (3.7), and shoot length (30 mm) were from a medium containing MS supplemented with 10 mg·L−1 BAP, 15 mg·L−1 NAA, and 1.5 mg·L−1 zeatin. Frequency of root formation and survival of regenerated plants were greater on medium containing half-strength MS salt than medium fortified with full-strength MS salt. The best frequency of root formation (average 87.4%) and survival of regenerated plants (average 95.4%) were from media containing 6.0 mg·L−1 zeatin and 1.0–1.5 mg·L−1 IBA and half-strength MS salt. Micropropagated shoots with well-developed roots were acclimatized under greenhouse conditions in pots containing rich sandy loam soil (pH = 7.2). The RAPD amplification products were monomorphic in micropropagated plants and similar to those of the mother plant. No polymorphism was detected, indicating genetic uniformity of micropropagated plants. This is the first report of micropropagation of C. crepidioides, which will facilitate in vitro mass propagation, conservation, and germplasm exchange of this endangered African vegetable.

In Vitro Regeneration of Endangered Medicinal Plant Heliotropium kotschyi (Ramram).

Heliotropium kotschyi (Ramram) is an important endangered medicinal plant distributed in the Kingdom of Bahrain. Plant tissue culture technique is applied for ex situ conservation study. Nodal stem segments are cultured in modified MS media supplemented with various combination and concentration of plant growth regulators (PGRs). Plants are regenerated via shoot organogenesis from the nodal meristems. Plants are regenerated in three different steps: initial shoot development, shoot multiplication, and rooting. After 4 weeks of culture, 100 % explants respond to shoot initiation on the medium containing 8.88 μM BAP and 5.71 μM IAA. The highest frequency of shoot regeneration is observed in the same media after second subculture of shoots. The highest rooting frequency is observed in the presence of 2.85 μM IAA. After root development, the plantlets are transferred to pots filled with soil and 60 % of plants survived after 45 days. This plant regeneration protocol is of great value for rapid desert plant propagation program.

BREAKING UP DORMANCY OF ADANSONIA DIGITATA L. SEEDS AND REGENERATION OF PLANTLETS FROM STEM NODAL SEGMENTS IN VITRO

In Yemen, there are only three trees of Adansonia digitata L. which is adopted in this study. Natural regeneration and independent germination of Adansonia digitata is poor although it is one of the tallest trees in the world that was recorded to live about 4000 years. Unfortunately, the tree is a subject of extinction. In the present study, the seeds of Adansonia digitata L. were pretreated with full and half strength of hydrochloric, sulfuric and nitric acids for one and/or two hour as a mean of scarificationtobreakthedormancyoftheseeds.Seedswerethensurfacesterilizedandallowedtogerminateonhormone–freeMS basal salts culture medium in vitro. The obtained seedlings were used as a source of nodal segments explants for propagation in vitro. Results of the present study have shown that all of the acid treatments overcame seed dormancy and enhanced seed germination in various degrees. The percentage of seed germination increased from 18.6% in the control group to 86.6% using a pretreatment of half strength sulfuric acid for one hour. The germinated seeds grown into seedlings of a mean of 11.1 centimeter after 8 weeks of growth. Stem nodal segments were cultured on agar solidifiedMSbasalsaltssupplementedwithvarious concentrations of benzyle adenine (BA) and naphthalene acetic acid (NAA). The maximum regenerant shoot length (15.1±03 cm) was obtained with 8 mg/l BA plus 0.5 mg/l NAA while The maximum number of leaves per regenernt reached up (7.2±0.2) using 10 mg/l BA + 1.0 mg/l NAA.The obtained regenerants developed roots on the same culture media. They were acclimatized for one week and then transferred to soil.

Radiossensibilidade de sementes e segmentos caulinares de maracujazeiro-amarelo submetidos à radiação gama

Os objetivos neste estudo foram avaliar os efeitos da radiação γ sobre a sobrevivência e desenvolvimento de gemas de segmentos caulinares e germinação de sementes de maracujazeiro-amarelo (Passiflora edulis f. flavicarpa) in vitro. Os explantes de segmentos caulinares (2cm) contendo uma gema axilar, obtidos a partir de plântulas provenientes da germinação in vitro de sementes de maracujá, foram tratados com raios γ em dosagens variando de 10 a 60Gy e subsequentemente transferidos para meio Murashige e Skoog (MS), suplementado com 4,0mg L-1BAP. Para a irradiação das sementes, foram testadas doses que variaram de 75-200Gy e a germinação e sobrevivência das plântulas foram avaliadas após 30 e 60 dias da inoculação em meio MS, respectivamente. Após 30 dias da irradiação de segmentos caulinares, observou-se redução de 50% na sobrevivência dos explantes e no número de brotações formadas in vitro nas doses de 21,31 e 12,20Gy, respectivamente. A DL50 para a germinação de sementes é 160Gy, enquanto que, para sobrevivência das plântulas, é de 125Gy.

A high-frequency in vitro multiplication, micromorphological studies and ex vitro rooting of Cadaba fruticosa (L.) Druce (Bahuguni): a multipurpose endangered medicinal shrub.

An efficient and reproducible in vitro propagation protocol has been established for Cadaba fruticosa (L.) Druce. Surface-sterilized nodal stem segments of mature plant were used as explants for culture establishment. Multiple shoots were optimally differentiated from the nodal stem explants through bud breaking on Murashige and Skoog (1962) medium containing 3.0mg l(-1) benzyladenine (BA). The effect of different plant growth regulators and minerals were studied on different stages of micropropagation procedure (i.e., explant establishment, shoot multiplication/growth and ex vitro rooting). Additionally, for enhancing shoot multiplication during subculture, MS medium was modified (MMS) with higher levels of magnesium, potassium and sulphate ions. Out of these, MMS3 medium containing 0.25mg l(-1) each of BA and Kin (N6-furfuryladenine), with 0.1mg l(-1) NAA (α-naphthalene acetic acid) was found the best for shoot multiplication (42.45 ± 3.82 per culture vessel). The in vitro regenerated shoots were rooted under ex vitro conditions on treating the shoot base with 500mg l(-1) of IBA (indole-3 butyric acid) for 3min on sterile Soilrite®. The ex vitro rooted plants were hardened in the greenhouse and transferred to the field with ≈85% survival rate. There were not any visual differences between wild and micropropagated plants in the field, although the later underwent significant changes during acclimatization. Micromorphological changes on leaf surface characters from in vitro to acclimatized plantlets were studied in terms of development of glandular trichomes, changes in vein spacing and vein structure in order to understand the nature of plant responses towards environmental conditions. The method developed and defined can be applied for commercial cultivation, which may be important for extraction of bioactive compounds and may facilitate conservation of this multipurpose endangered medicinal shrub.