Callus Initiation Research Articles

Introduction to in vitro culture and callus initiation in Salvia hispanica L. (chia)

Aim. Preparation of aseptic seedlings Salvia hispanica L., callus initiation in vitro and establishment of primary explants suitable for the callus production. Methods. Seeds are sprouted on our own modification of conventional methods. The non-hormonal Murashige-Skoog agarized nutrient medium was used as basic medium for the experiments. Parts of one-month seedlings (roots, hypocotyl, cotyledon leaves) were used as explants for the use of the colza. We added growth regulators (BAP, 2,4-D) in different concentration combinations into the nutrient medium for callus initiation. Statistical processing was performed in Microsoft Office Excel. Results. Aseptic S. hispanica seedlings have been obtained. The callus growth was initiated on all types of explants, the dependence of the callus intensity on the type of explants and the growth regulators content in the nutrient medium was established. Morphogenic callus and root-regenerants have been obtained. Conclusions. Hypocotyl was the most suitable primary explant for callus growth. Seedlings, leaves and roots showed low morphogenetic capacity. The nutrient medium with an elevated 2,4-D content was the most effective for initiation of callus genesis and proliferation of non-morphogenous callus. A high concentration of 2,4-D in the medium improves S. hispanica callus growth but suppresses its morphogenic ability.Keywords: Salvia hispanica (Chia), in vitro culture, callus.

Elicitors Enhancing Phenolics Content and Related Gene Expression Variation in Petal - Derived Calli of Rosa damascena Mill.

DUE to increasing importance of roses in producing secondary products including essential oils and pharmaceutical compounds as phenolics, several protocols were developed from different explants of roses. For that purpose, an efficient callus initiation procedure for Rosa damascena Mill. cultivated in Taif, KSA was developed by using petal explant cultures supplemented with nine elicitors for improving phenolics content. As well, related genetic expression variation of the elicited calli was studied by biochemical (protein and isozyme) analyses. Except for rose and geranium oils, seven elicitors increased the phenolics content in R. damascena calli compared with the control. High qualitative differences were observed in their banding patterns reflecting biochemical aberrations in treated calli. A dendrogram, based on UPGMA method of cluster analysis, upheld the notable fluctuations in phenolics content and gene expression occurred by different elicitors.

A rapid in vitro protocol for callus production in Piper aduncum L. propagation of East Kalimantan supplemented with gradient sucrose solutions

Piper aduncum L. is a medicinal plant which contains important active compounds such as phenyl propanoids, lignoids and flavonoids. Furthermore, it is one of larvicidal agent against mosquitoes Aedes aegypti L. Alternative use of in vitro callus P. aduncum L. are as a substitute for medicinal raw materials and becomes one way to maintain the sustainability of germ plasm utility, which is related to its difficulties in conventional cultivation. An experiment based on saline formulation of Murashige and Skoog (MS) and Wood Plant Medium (WPM) combined with different 2,4-D and kinetin concentration was evaluated. Several conditions without plant growth regulator hormones with nodule segment explants produced good multiplication process. Inoculated micro-cutting segments of nodules were grown in media with an additional 2 mg/L of 2,4-D, 2 mg/L of kinetin and 30 g sugar produced multiplication of clear shoots and callus formation and were developed after 4 weeks of planting. Additional treatment of 60, 90, 120 g sugar showed slow growth as indicated by callus formation after 6-8 weeks of planting. Treatment of 30 g sugar showed better growth than other treatments, which triggered the survival of callus growth until 12 weeks after planting. The other treatments showed cell death after callus initiation due to browning process.

SHOOT TIPS AND NODES MULTIPLICATION, CALLUS INTIATION AND DIFFERENTIATION OF CALENDULA OFFICINALIS L. CULTURE IN VITRO

This investigation was carried out in the laboratory of cells and plant tissue culture at department of Horticulture and Landscape Design,College of Agriculture and Forestry,Mosul university,to propagate Calendula officinalis L. cv. Lemon Queen in vitro by multiplication shoot tips and nodes taken from field culture on MS medium supplemented with (0.0,0.5, 1.0, 1.5, 2.0) mg /l BA,shoot produced in vitro cultured on MS medium supplemented with (0.0,0.5, 1.0, 1.5, 2.0) mg/l IBA,moreover parts of leaves had been cultured on MS medium supplemented with 0.1 mg /l 2,4-D for callus initiation, callus cultured on MS medium supplemented with (0.0, 0.1, 0.5, 1.0) mg/l BA interacted with(0.0, 0.1, 0.5, 1.0) mg/l IBA for differentiation. Data refer that best multiplication 7.8 shoots/explants with 2.7 cm length were obtained from cultured shoot tip drived from field cultured on MS medium supplemented with 1.5 mg/l BA after eight weeks compared with nodes which gave 3.6 shoots / explants with 2.8 cm length from cultured on MS medium supplemented with 1.0 mg/l BA after eight weeks, highest rooting 70% with highest roots number 8.8 obtained from shoot tips produced in vitro cultured on MS medium supplemented with 0.5 mg /l IBA, highest fresh weight of callus 15.9 gm was initiated from callus cultured on MS medium supplemented with 1.0 mg /l IBA or from callus cultured on medium supplemented with 0.5 mg/l BA and 1.0 mg /l IBA, 4.0 shoot /explant regenerated from callus cultured on MS medium supplemented with 0.5 mg/l BA and 0.5 mg/l IBA, 10.2 root /explant obtained from callus cultured on MS medium supplemented with 0.1 mg /l BA after eight weeks.

In vitro regeneration of double haploid line of African marigold (Tagetes erecta) derived from ovule culture using non-axillary explants

An efficient protocol has been developed for in vitro regeneration of double haploid line of African marigold variety Local Orange derived through ovule culture. Maximum survival (85%) of leaf explant was found in the treatment comprising of Bavistin (0.2%), Ridomil (0.2%) and 8-HQC (200 ppm). The surface sterilization treatment of explants with mercuric chloride (0.1%) for 3 minutes resulted in maximum survival (88.33%) and minimum contamination (18.33%). Three different portions of leaf explant (Tip, middle and basal) were used for regeneration. The treatment comprising MS medium supplemented with BAP (0.5 mg/l) and NAA (0.25 mg/l) resulted in maximum percent regeneration (86.25), lowest number of days taken for callus initiation (3.05), minimum number of days for shoot regeneration (13.25), maximum number of micro shoots per explant (3.50), minimum number of days to reach the stage of sub culturing (5.75) with basal portion of leaf explant. Among the different explants, basal portion of the leaf was found as the best explant for maximum regeneration. The basal portion of the leaf explant was further treated with best regeneration treatment, i. e. BAP (0.5 mg/l) and NAA (0.25 mg/l) along with different concentrations of putrescine and it was observed that maximum percent regeneration (89.83%), minimum number of days for shoot regeneration (13.50), maximum number of micro shoots per explant (6.25) and minimum number of days to reach the stage of subculturing (5.75) were found in the treatment combination of BAP (0.5 mg/l), NAA (0.25 mg/l) and Putrescine (50 mg/l).

Efficient and reproducible somatic embryogenesis and micropropagation in tomato via novel structures - Rhizoid Tubers.

A dual in vitro regeneration system consisting of indirect organogenesis and somatic embryogenesis (SE), applicable to several varieties of tomato—Solanum lycopersicum (cv. Riogrande, cv. Roma, hybrid 17905 and model cv. M82) has been established. This system is both improved and highly reproducible compared to current methods. Callus initiation, plant regeneration and SE was developed for one-week-old cotyledon explants. Indirect organogenesis via callus induction (CI) was developed for all four varieties of tomato used in this study. One-week-old tomato seedlings were used as a source of cotyledon and hypocotyl segments as explants. The explants were subsequently cultured on Murashige and Skoog (MS) medium supplemented with different combination and concentrations of plant growth regulators (PGRs). Substantial trends in regeneration and propagation response were observed among the varieties and treatments. For commercial varieties cvs. Riogrande and Roma, maximum CI was observed at 2 weeks in CIMT9 (0.5 mg/L NAA, 1 mg/L BAP) and CIMT12 (2 mg/L IAA, 2 mg/L NAA, 2 mg/L BAP, 4 mg/L KIN). However, cv. M82 responded after 4 weeks to a combination of treatments CIMT9 (0.5 mg/L NAA + 1 mg/L BAP) and CIMT13 (2 mg/L IAA + 2 mg/L NAA + 2 mg/L BAP + 4 mg/L ZEA) for the production of calli. Subsequent shoot and root organogenesis were optimized for all four varieties. Cv. Riogrande, exhibited fastidious in vitro regeneration potential and selected for induction of somatic embryos via SE involving novel structure: rhizoid tubers (RTBs). Numerous fine hair like rhizoids (~23/explants) were first developed from cotyledon and hypocotyl explants cultured on MS medium supplemented with 0.5 or 2 mg/L NAA at pH 4.0 in dark conditions. Further incubation of each rhizoid under light conditions on MS media supplemented with 5 mg/L TDZ or BAP at pH 4.0 led to the formation of a novel structure—rhizoid Tubers (RTBs). Thus, as evident from histology, SE in Riogrande tomato species requires a medium with pH of (4.0) and higher concentration of cytokinins (BAP/TDZ) to form on average 40–45 RTBs from both explants. Histological and morphological studies revealed that RTBs develop through different stages of embryogenesis to multiple plantlets, on MS medium with 5 mg/L TDZ/BAP at normal pH (5.8). The results obtained indicated that the induced somatic embryos of tomato with lower pH are a more efficient mode of propagation than the organogenesis with or without callus formation. The RTBs led to a complete plantlets regeneration in 45 days compared to indirect organogenesis at 60 days.

Effect of combination explant difference leaf part and concentration of active charcoal on callus initiation mangrove (Rhizophora Apiculata BI) by in-vitro

Rhizophora Apiculata BI is one of the mangroves that has many benefits, but to cultivate naturally takes a long time. The alternative to such problems is culture cultivation in vitro. One stage of in vitro culture is through callus formation. Initiation of callus in mangrove plants is inhibited because explant release phenol compound that causes browning. Thus, it takes explant and charcoal active in a medium that can support growth explant. The purpose of this research is to study the effect of the combination of leaf explant and different activated charcoal concentration on callus initiation, to analyze the color and texture of callus produced, and to study the effect of the combination of leaf explant and different concentration of activated charcoal on the growth of explant. The basal medium used was Murashige and Skoog (MS) formula with the addition of NAA 1 mg/L and BAP 0.3 mg/L, and activated charcoal at concentration 4, 12, 20 g/L as the medium treatments. A different section of explant between base and tip of leaves combined with active charcoal were grown in MS medium. Each treatment was conducted three replications. The results showed that the combination of explant section, i.e. base of the leaf as well as activated charcoal with a concentration of 12 g / L could induce callus formation. The color and texture of callus were brownish yellow and compact. The best growth (0.1685 g) of callus was obtained at the leaf tip section combined with 4 g / L activated charcoal.

Identification of exudates from callus of Mangrove Plant (Rhizophora apiculata BI) in vitro.

Rhizophora apiculata is species in mangrove vegetation. The mangroves leaves were the water-storage type. Induction of callus from the leaf of R. apiculata faced problems, i.e., browning phenomena and exudates. This research aimed to evaluate the production of exudates and their correlation with the browning of explant and to identify kind of compounds of exudates. The leaf that is used as explants was divided into the base and tip section was grown in Murashige and Skoog (MS) medium with the addition of NAA 1 mg/L, BAP 0,3 mg/L, and activated charcoal 12 g / L. The treatments included 24 hours placed in the dark and 24 hours placed under light, 16 hours placed in the dark and 8 hours under the light, 8 hours placed in the dark and 16 hours under the light; the treatments were repeated four times. Respond of explants were descriptively observed, while the exudates were analyzed by Benedict test. The results showed that light treatment could induce callus formation while 24-hour dark treatment could reduce browning. The novelty of this research lies upon the process of browning prevention using light duration treatment. Therefore, browning could be prevented, and mangrove culture could successfully produce callus. Not at all explants produced the exudates. Light treatment can be used to avoid exudates production. Some of the exudates consisted of carbohydrates. Nevertheless, another one consisted of a salt solution. These exudates made a disturbance in callus initiation of R. apiculata.

Establishment of Punica granatum L. peel cell culture to produce bioactive compounds

The pomegranate (Punica granatum L.) fruit harbors remarkable health-beneficial properties. Most of its healthy secondary metabolites are located in their peels. Therefore, pomegranate peels (PPs) can be used to produce high-value healthy compounds. PP cell cultures may also be an attractive alternative source since it can be established throughout the year regardless of seasonal effects. The aim of this study was to establish a novel PP cell culture for future research and biotechnological applications. We first established procedures to sterilize and control oxidative browning of the peel explants. We found that surface sterilizing with 3% sodium hypochlorite for 15 min, frequent sub-culturing of the explants on media containing polyvinylpyrrolidone plus silver nitrate was the optimal treatment for oxidative browning control and callus initiation. We also found that 6-benzylaminopurine (BAP) induced callus initiation, whereas 3-indoleacetic acid (IAA) repressed it. The callus daily growth rate was maintained at 1% regardless of the various growth media examined. Moreover, our results show that although there was a minor effect of the growth media on polyphenol accumulation and antioxidant activity, the exposure to light and high sucrose levels led to higher levels (up to 205 times than the control) of secondary metabolites such as anthocyanins, ellagic acid, punicalagin and gallic acid. In summary, this work laid the foundations for further research that will be needed to study and commercialize PP cell cultures. In this present study, we addresses the establishment of pomegranate peel cell cultures for future research and biotechnological applications, such as production of health-beneficial secondary metabolites.

Effect of plant growth regulators on plant regeneration of hyacinth bean (Lablab purpureus (L.) Sweet) through nodal explants

Nodal explants of three accessions namely BD-101, BD-122 and BD-8001 of hyacinth bean (Lablab purpureus (L.) Sweet) were cultured for four weeks on MS medium supplemented with different concentrations and combinations of NAA (0, 0.1 mgl-1) and BAP (0.5, 1.0 and 2.5 mgl-1) for plant regeneration. The highest shoot initiation was observed in 0.5 mgl-1 BAP while the lowest shoot initiation was found in 2.5 mgl-1 BAP with 0.1 mgl-1 NAA. Earlier shoot initiation was exhibited in 0.5 mgl-1 BAP with 0.1 mgl-1 NAA. The highest number of leaflets and higher shoot lengths were observed in MS medium. Comparatively higher number of shoots was found in BAP (0.5-2.5 mgl-1). The highest percentage of callus was initiated in medium supplemented with 1.0 mgl-1 BAP. Earlier callus initiation and larger callus size were found in combination of BAP (0.5-2.5 mgl-1) with 0.1 mgl-1 NAA. BD-122 cultured in MS medium was found superior for shoot regeneration through node culture. Four different concentrations of IBA (0.1-1.5 mgl-1) were used for rooting. The highest percentage (86.67 %) of rooting was found in MS medium containing 0.1 mgl-1 IBA at four weeks. Rooting frequency decreased with the increasing concentration of IBA. The accession BD-8001 had 99.60 % rooting in 0.5 mgl-1 IBA. The highest number of longest roots was exhibited in 0.1 mgl-1 IBA. The regeneration protocol developed from nodal explants has applicability in improvement of hyacinth bean.
 Bangladesh J. Agril. Res. 44(1): 27-42, March 2019

Effect of nitrogen sources and 2, 4-D treatment on indirect regeneration of ginger (Zingiber officinale Rosc.) using leaf base explants

Ginger is an important monocotyledonous plant belonging to the family Zingiberaceae. The objective of this study was to investigate the regeneration potential of ginger using leaf base explants. Auxins such as 2, 4-D and NAA in combination with BA were used for initiation of callus. Different combinations of both ammonium (NH4+) and nitrate (NO3-) were also studied for efficient callus production. High frequency of white friable calli was observed on modified Murashige and Skoog (MS) medium supplemented with 2.0 mg/L 2, 4-D, 0.5 mg/L NAA and 0.5 mg/L BA. The highest shoot induction (92.33%), shootlets number (7.33±0.33) and length (88.33±4.40) mm were achieved on MS media containing 0.5 mg/L BA. Regenerated shoots were transferred to in vitro rooting media containing 1.0 mg/L IBA. Afterwards, plantlets with well-developed root and shoot system were subjected to a twostep hardening process. 71% of plantlets survived after secondary hardening without any abnormal morphology.

Histological Investigations During <i>In-Vitro</i> Adventitious Shoot Regeneration in Leaflet Explants from Nodal Segments of a Mature Tree of <i>Albizia procera</i> Benth.

This is a 60 day study of transformation from leaf to callus and adventitious shoot regeneration. For initial 30 days leaf explants were kept on MS medium supplemented with 3% sucrose, 2.5 μM 2,4-D and followed by a subsequent adventitious shoot differentiation phase for another 30 days on half MS medium supplemented with 0.25 μM each of BA and IBA. The histological investigation revealed sequential array of events starting from initiation of callus on upper and lower epidermis of inoculated leaflet to initiation of green shoots from the mass of callus in the culture medium.

Use an Organic Biostimulant (Vermicompost Tea) For Enhancement In Vıtro Callus Growth in Sainfoin (Onobrychis viciifolia Scop.)

The health and vitality of callus growth is one of the prerequisites for the success of further in vitro studies. This study investigated the efficiency of different percentage (0%, 10%, 20%, 30%, and 40%) of vermicompost tea as an organic substance on in vitro callus growth in sainfoin. Morpho-physiological responses of calli to vermicompost tea measured under in vitro conditions. As a result of this investigation, a combination of plant growth regulators (4 mg/l BAP and 0.5 mg/l NAA) with 20% of vermicompost tea causing significant callus initiation and growth in sainfoin stem explants. Under the light of these scientific findings, vermicompost tea might be used as an organic bio stimulant for efficient callus growth and complementary to commercial chemical hormones in sainfoin. This research is important due to it can contribute positively to the plant species that are difficult in terms of callus growth and plant regeneration in tissue culture.

Micropropagation of Little Bluestem (Schizachyrium scoparium L.)

Native grasses are increasingly used in the landscape. Little bluestem (Schizachyrium scoparium L.), a perennial bunchgrass native to most of the United States, has ornamental traits, such as variation in leaf color, differences in growth morphology, and attractive seed heads. Traditionally, cultivars of little bluestem are propagated by division, which limits the production of new plants. Our objective in this study was to develop an improved micropropagation protocol for little bluestem that would produce true-to-type plants. In 2016, we cultured immature inflorescences of eight genotypes of little bluestem on Murashige and Skoog (MS) medium with four combinations of kinetin (1.0 or 2.0 mg·L−1) and 2,4-D (0.5 or 1.0 mg·L−1) under three levels of light (dark, semilight, full light) to initiate callus. Cultures were evaluated 30 days after initiation and those that had initiated callus were subcultured. Media for subculturing and rooting contained either 0.1 mg·L−1 or no 1-Naphthaleneacetic acid (NAA). Light level had no effect on callus initiation. Initiation media with 1.0 mg·L−1 kinetin and either level of 2,4-D induced callus at almost twice the rate of media with 2.0 mg·L−1 kinetin, and cultures initiated on those media also produced almost twice the number of rooted plants over all genotypes. Genotype affected the number of rooted plants produced. The addition of NAA to medium for subculturing and rooting did not increase the number of rooted plants. In 2017, we cultured immature inflorescences of four genotypes of little bluestem on MS medium with 0.5 mg·L−1 2,4-D and either 1.0 mg·L−1 kinetin or 6-benzylaminopurine (BAP) under full light. Cultures were evaluated 30 days after initiation. Cultures that had initiated callus were subcultured onto MS medium with the same growth regulators as the initiation medium but without 2,4-D. Cultures were cycled between subculture medium with growth regulator and subculture medium with no additional growth regulator until rooted. Cultures initiated and subcultured on medium with BAP initiated two to three times more callus than those on kinetin and produced twice as many rooted plants. Our recommendation for rapid micropropagation of little bluestem is to initiate cultures on MS medium with 1.0 mg·L−1 BAP and 0.5 mg·L−1 2,4-D. After callus initiation, cultures should be subcultured to medium with BAP but no 2,4-D, alternating with medium with no additional growth regulators, until rooted.

High-efficiency somatic embryogenesis of a broad range of Brazilian Saccharum spp. hybrids (sugarcane) varieties using explants from previously established in vitro plants

The current study assessed the embryogenic potential of different Brazilian Saccharum spp. hybrid (sugarcane) varieties, using explants from previously established in vitro plants, and determined the morphogenic capacity of calluses to regenerate plants during consecutive subcultures. After examining various explant lengths (1 to 15 mm) for callus initiation, an optimal length of 12 mm was determined, and 14 sugarcane varieties were studied. Callus induction occurred on Murashige and Skoog medium with 3.0 mg L−1 2,4-dichlorophenoxyacetic acid, in the dark. After 40 d, calluses were divided according to their predominant type: the mucilaginous part remained in the dark on fresh induction medium, whereas the compact nodular callus fraction was transferred to a regeneration medium containing 1.86 mg L−1 1-naphthaleneacetic acid and 0.09 mg L−1 6-benzylaminopurine and was cultivated in light. This callus selection based on morphological type took place over three consecutive subcultures, spanning 22 wk. The embryogenic process was asynchronous and the formation of the first plants was registered at 6 wk of cultivation. The embryogenic callus regeneration rate was kept at over 60% until the third subculture in 13 of the 14 varieties examined. However, delayed regeneration was observed, likely due to the aging of calluses. The protocol reported here utilized the embryogenic potential of sugarcane varieties in combination with efficient and quick regeneration and should thus provide an attractive alternative source material for the production of transgenics and/or large-scale clonal multiplication of sugarcane.

Indirect shoot organogenesis of a valuable medicinal plant Paederia foetida L. using nodal explants

Paederia foetida L. is an important medicinal herb harboring lots of essential drug producing metabolites and the plant has been going to be endangered due to lack of proper strategies for conservation. Since, indirect organogenesis by tissue culture is considered as the valuable tools for rapid multiplication and improvement of plant genetic resources, application of this technique should be very imperative for the conservation of this valuable and rare medicinal plant. Hence, the research effort was made to develop a suitable protocol for indirect organogenesis in-vitro using nodal explants of P. foetida. After surface sterilization, the explants were submitted to MS medium supplemented with different concentrations and combinations of plant growth regulators for showing the performance in terms of callus induction, shoot proliferation and root initiation. Among the surface sterilants used, 0.1% HgCl2 treated for 2 minutes and 3% NaOCl treated for 10 minutes showed better performance and maintained 100% and 80% survivability respectively. MS medium supplemented with 2, 4-D at 1.5 mg L-1 showed better performance than others in terms of initiation of callus from nodal explants. In contrast, NAA at 0.5 mg L-1 and BAP 0.2 mg L-1 showed highest rate of callus proliferation with somatic embryos from proliferated callus. During shoot organogenesis, MS medium supplemented with BAP 2 mg L-1 showed better results for the regeneration of shoots from embryogenic calli. The shoots derived from callus produced roots by half strength MS medium supplemented with IAA. After acclimatization, the plantlets were allowed to ambient condition for further establishment. Our findings claim the establishment of a suitable protocol for indirect organogenesis of P. foetida that could be employed for rapid multiplication, conservation and sustainable utilization of P. foetida as valuable genetic resource.

In vitro plant regeneration of wild eggplant (Solanum sisymbriifolium) to produce large number of rootstocks for tomato grafting

The experiment was conducted to develop a suitable protocol for high frequency plant regeneration of wild eggplant (Solanum sisymbriifolium) in order to produce a large number of rootstocks for tomato grafting for the management of wilt disease. To obtain in vitro seedlings of S. sisymbriifolium, seeds were treated with various concentrations of GA3 (Gibberellic acid) prior to place them in germination media (½ strength MS) and 750 mg/L GA3 was found as a suitable concentration resulting the highest (76.67%) germination rate. Various factors namely combination of plant growth regulators, explant types and explant age were investigated for development of an efficient plant regeneration system of S. sisymbriifolium. Cotyledon and hypocotyl explants of S. sisymbriifolium were cultured on MS medium supplemented with various concentrations of BA (6-Benzylaminopurine), NAA (α-Naphthalene acetic acid) and 2,4-D (2,4-Dichlorophenoxy acetic acid), to determine suitable medium for callus and shoot initiation. Fourteen days old cotyledon explants were found more responsive than that of hypocotyl, both in callus and shoot induction. The highest callus initiation (100%) and shoot regeneration (73.33%) were observed in MS media supplemented with 0.5 mg/L NAA + 1.0 mg/L BA and 0.2 mg/L NAA + 3.0 mg/L BA, respectively. MS medium supplemented with 0.1 mg/L NAA showed the highest frequency (86.67%) of rooting. The regenerated plantlets were acclimatized in pot soil and eventually used as rootstock for tomato (Solanum lycopersicum cv. BARI hybrid 4) grafting. The grafted plants showed no wilt disease in field condition until maturity.

Regeneration of White Lupin, Lupinus albus, Plants form Hypocotyl Stems

This study succeeded in regenerating the white lupin, Lupinus albus, plants from differentiated the hypocotyl stems callus of its sterilized seedlings. The results indicated to superior the solid Murashige and Skoog (MS) medium provided with 1.0 mg L-1 NAA in callus initiation with 90% after 20 days of culture from the other of selected media. The rate of the value of the callus fresh weight was 3.3 gm after 40 days of growth, which gives indication on the viability and activity of callus. The continued of callus subculture on the same its initiated medium led to spontaneous production of 22 shoots. These regenerated shoots rooted when cultivated in MSO with its full strength at ratio 50% after 26 days, producing the whole lupin plants in the culture media. .

Evaluation of In Vitro Germination and Callus Induction for Antimicrobial Activities of Jatropha curcas L.

There are many problems related to seed germination and in vitro cultivation of Jatrophacurcas L, this research was to solve this obstacle. This study also aimed to use triphenyl tetrazoliumchloride (TTC) as a new substance for in vitro callus initiation and growth in plant tissue culture fieldsfor the first time. In addition, an efficient regeneration method via seed culture has been developed.For this purpose, different media (MS; B5; WPM with B5 vitamins; wetted cotton and water) were usedfor seed germination. High seed germination percentage for all treatments were achieved after 24days from incubation. WPM was effective medium in increasing the total length of seedlings. Themaximum dry weight (0.24 g) of Jatropha curcas shoots was recorded with B5 medium. In vitro shootformation, MS medium supplanted with 2.0 μM IBA +10 μM BA and MS medium+2.5 μM BA+2.5 μMNAA recorded the highest shoot formation percentage (100%) after one month. Concerning callusinduction, data implied that different growth regulators tested (TDZ, BA, KIN) and TTC as a newsubstance used in plant tissue culture could induce the formation of callus in cotyledons of J. curcasseedlings, furthermore, the combination of BA and Kin in various concentrations was an appropriatemedium for inducing the formation of callus and promoting its growth. Also, callus formation andgrowth were significantly affected by explant types. The balance between auxin and cytokinin is thelimited factor for callus induction. MS nutrient medium supplemented with 2,4-D or NAA or both helpedto form callus at high percentage. regarding antimicrobial activity, the antimicrobial potential ofdifferent plant extracts was screened against four pathogenic microorganisms and reference bacterialstrains, the ethanolic extract of the roots was found to show greater inhibition of B. cereus and E. coliwith zones of inhibition, 21.7 and 10.4 mm respectively, when comparing the extracts from in vivoplant and in vitro callus. Also, ethanol extract from the leaf of the mother plant showed higher inhibitoryzone than that of the leaf-derived callus against S. aureus, B. cereus and E. coli. On the other hand,butanol extract of hypocotyl-derived callus was achieved the highest fraction effect resulted fromdifferent fractionation of crude methanolic extract against antibacterial activity of E. coli, followed byethyl acetate- fraction (15.0 and 13.5 mm) respectively, in contrast, water- fraction came in the last.

High frequency regeneration from leaf derived callus and its histological studies in Lepianthes umbellata

ABSTRACTA protocol was standardized for callus initiation and shoots regeneration from leaf explants of Lepianthes umbellata. The explant was cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of 2,4-Dichlorophenoxyacetic acid (2,4-D), 6-Benzylaminopurine (BAP), and 1-Naphthalene acetic acid (NAA) for shoot bud initiation and multiplication. High frequency of shoot regeneration and maximum number of shoots per explant were achieved on MS medium fortified with 0.5 mg L−1 NAA and 1.0 mg L−1 BAP. The elongations of shoots were achieved on the same medium supplemented with 1.0 mg L−1 Gibberelic acid (GA3), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA) at different concentrations on half strength MS medium were used for rooting of the micro shorts. Highest efficiency for rooting was observed on MS with 1.5 mg L−1 IBA. Regenerated plantlets were successfully transferred for acclimatization (70%). Histological studies showed that embryoids and shoot buds initiating from the callus masses, that is, indirect organogenesis. Cytological variations were observed in callus cultures.