MS Salts Research Articles

Evaluation of responses embryogenic Cycas revoluta thumb., from callus culture obtained in vitro

Processes cell differentiation and dedifferentiation are included in the development of biotechnology protocols to promote somatic embryogenesis as an alternative to the in vitro propagation of plants, somatic embryos may be an excellent strategy for both propagation and conservation of fossil species such as cycads. They were evaluated in vitro with different strategies, morphogenic responses associated with obtaining somatic embryos of C. revolute. calli of megagametophytes, subsequently subcultured in four combinations of basic salts of Murashige and Skoog (1962) MS, with the addition of benzyladenine (BA), and 2, dichlorophenoxyacetic (2,4-D) were used, and kinetin (K), and picloram. In the results, It was possible to characterize potentially embryogenic callus, evaluating the levels of both cellular differentiation, necrosis, texture and color; and increases mass or weight considered to start differentiation or proembryogenic or globular type. Calli were subcultured in a medium containing MS salts, incorporating abscisic acid (ABA) in 0, 0.38, 1.13, 3.78 and 5.67 uM doses influenced both the production and maturation of somatic embryos. Embryonic structures, presented a pink coloration characteristic strongly associated towards maturity. The effect of combinations of BA, Kin, 2,4-D, GA3 and ANA influenced the development and germination of mature somatic embryos. And the combination of 1.36 mM 2,4-D+4.44 uM BA promoted the appearance of calluses with a compact texture, characteristic related to their embryogenic potential. The purpose of this research in Cycas sp was to contribute to the study of the in vitro morphogenic responses of this group of plants. And somatic embryogenesis, will allow the obtaining and multiplication as well as its preservation of Cycas sp. Gender that is evolutionary very important

Micropropagation and Somatic Embryogenesis Induction of Gardenia jasminoides Plants

Gardenia jasminoides Ellis is an evergreen plant with fragrant creamy white flowers and glossy, darkgreen leaves, belongs to family Rubiaceae and member of the genus Gardenia. The current study aimed to afford a new technique for in vitro propagation and callus induction of G. jasminoides by using single nodes excised from soft cuttings using full strength MS salts supplemented with 30g/l sucrose, 4g/l gelrite and different concentrations of plant growth regulators. Generally, the current study showed that NAA and BA at 0.0 and 1.0 mg/l, respectively gave rise to the best results for initiation stage. Meanwhile, fortified the basal MS medium with BA and NAA at 2.0 and 0.250 mg/l, consecutively; resulted in the best results with respect to multiplication stage. Regarding rhizogenesis, the neoformed shoots produced from vegetative multiplication stage were rooted successfully upon cultured on MS medium supplemented with 1.0 mg/l IBA and NAA at either 0.250 or 0.500 mg/l, whereas the highest number of roots was recorded besides callus induction. Regarding embryonic callus induction, adding 2,4-D at 1.0 mg/l; enhanced embryonic callogenesis. Neoformed plantlets were acclimatized ex vitro and in vivo vigorously in mixture of perlite and peatmoss at (1:1, v/v), which resulted in the highest percentage value of survival / percent (100%) and successfully flowered and showed true-to-type plants ex vitro.

Expression of wild rice Porteresia coarctata PcNHX1 antiporter gene (PcNHX1) in tobacco controlled by PcNHX1 promoter (PcNHX1p) confers Na+-specific hypocotyl elongation and stem-specific Na+ accumulation in transgenic tobacco

Soil salinization is a major abiotic stress condition that affects about half of global agricultural lands. Salinity leads to osmotic shock, ionic imbalance and/or toxicity and build-up of reactive oxygen species. Na⁺/H⁺ antiporters (NHXs) are integral membrane transporters that catalyze the electro-neutral exchange of K⁺/Na⁺ for H⁺ and are implicated in cell expansion, development, pH/ion homeostasis and salt tolerance. Porteresia coarctata is a salt secreting halophytic wild rice that thrives in the coastal-riverine interface. P. coarctata NHX1 (PcNHXI) expression is induced by salinity in P. coarctata roots and shows high sequence identity to Oryza sativa NHX1. PcNHX1 confers hygromycin and Li+ sensitivity and Na+ tolerance transport in a yeast strain lacking sodium transport systems. Additionally, transgenic PcNHX1 expressing tobacco seedlings (PcNHX1 promoter) show significant growth advantage under increasing concentrations of NaCl and MS salts. Etiolated PcNHX1 seedlings also exhibit significantly elongated hypocotyl lengths in 100 mM NaCl. PcNHX1 expression in transgenic tobacco roots increases under salinity, similar to expression in P. coarctata roots. Under incremental salinity, transgenic lines show reduction in leaf Na+, stem specific accumulation of Na+ and K+ (unaltered Na+/K+ ratios). PcNHX1 transgenic plants also show enhanced chlorophyll content and reduced malondialdehyde (MDA) production in leaves under salinity. The above data suggests that PcNHX1 overexpression (controlled by PcNHX1p) enhances stem specific accumulation of Na+, thereby protecting leaf tissues from salt induced injury.

High-frequency somatic embryogenesis, nuclear DNA estimation of milkweed species (Asclepias latifolia, A. speciosa, and A. subverticillata), and genome size stability of regenerants

A high-frequency somatic embryogenesis was developed for three Asclepias species, A. latifolia (broadleaf milkweed), A. speciosa (showy milkweed), and A. subverticillata (horsetail milkweed) using gibberellic (GA3) and the amino acid l-proline. A somatic embryo initiation medium consisting of MS salts (Murashige and Skoog, in Physiol Plant 15:473–497, 1962) with Gamborg’s (1968) vitamins, 1.5 µM 2,4-D, 2.3 µM kinetin, and 2% (w/v) sucrose supplemented with various concentrations of l-proline (0, 8.7, or 17.4 mM) combined with various of concentrations of GA3 (0, 2.9, or 5.8 µM), resulting in nine different media (MWM0–MWM8). All media produced callus, but no embryos were obtained on the control medium which contained no l-proline or GA3. Once calli produced somatic embryos, they were transferred to a medium referred to as somatic embryo conversion medium or SECM, which contained MS salts with Gamborg’s vitamins (Gamborg et al., Exp Cell Res 50:151–158, 1968), 2.3 µM kinetin, 2.9 mM GA3, 1.5% (w/v) sucrose, 8 g/L. The conversion percentage of somatic embryos into plants was high for all media, in particular for MWM2 (17.4 mM l-proline + 0 µM GA3) with conversion rates of 90.2, 93.4, and 97% for A. latifolia, A. speciosa, and A. subverticillata, respectively. Flow cytometry was used to estimate the nuclear DNA content of both seed-derived and in vitro grown plants. The 2C-DNA values of all three species were 0.92 pg, which did not differ from the values of in vitro grown plants, thus verifying that the regeneration system produces genetically stable plants.

Enhanced Alkaloid Production from Cell Culture System of Catharanthus roseus in Combined Effect of Nutrient Salts, Sucrose and Plant Growth Regulators

Callus and biomass culture of Catharanthus roseus L. were established to check for the presence of total alkaloid and its subsequent yield. Various treatments like strength of nutrient salts, sucrose concentrations and combinations of plant growth regulators (PGR’s) were applied to both MS and B5 in agar as well as suspension medium to test their effects on enhanced alkaloid content and its yield. There was no significant difference in any of the observable parameters of fresh wt, dry wt, alkaloid content, production, productivity and yield if the culture were treated similarly in both types of media formulations (MS or B5 salts). Physical state (agar solidified or the liquid suspension) of the medium had significant effect on all the parameters particular on fresh wt, alkaloid content and yields. Although, the fresh wt. and dry wt. of biomass in suspension culture was 2-3 times less than that of callus obtained from agar medium. However, the alkaloid content and yield was 2-3 times higher in suspension culture compared to agar medium in similar treatments. The highest alkaloid content observed was 5.67mg/g dwt in B5 suspension medium containing 3% sucrose and modified with 0.5mg/l 2,4-Dichlorophenoxy acetic acid (2,4-D) + 1 mg/l Kinetin (KIN) + 2mg/l α- naphthalene acetic acid (NAA). The combined effects of these factors on the enhanced production of total alkaloids were expected to contain higher yield of anticancer vinblastine and vincristine in the cell suspension culture system.

The effects of an osmoregulator, carbohydrates and polyol on maturation and germination of 'Golden THB' papaya somatic embryos.

This study evaluated the effect of osmoregulators and carbohydrates on the maturation and germination of somatic embryos of papaya 'Golden THB'. Cotyledon explants from papaya seedlings germinated in vitro on basal MS medium were cultured on somatic embryogenesis induction medium (IM) containing MS salts, myo-inositol, sucrose, agar and p-chlorophenoxyacetic acid. After 50 days, embryogenic calli were transferred onto maturation media (MM) for 45 additional days. For experiment 1, a MS-based medium supplemented with abscisic acid, activated charcoal and concentrations of PEG 6000 (0; 40; 50; 60 and 70 g L-1) was used, whereas for experiment 2 malt extract concentrations (0; 0.1; 0.2; 0.3 and 0.4 g L-1) were assessed. The normal cotyledonary somatic embryos produced in experiment 2 were transferred to the germination medium (GM). The GM consisted of full-strength MS medium, sucrose, agar and was supplemented with myo-inositol at varying concentrations (0; 0.275; 0.55 and 0.825 mM). The PEG concentrations tested impaired the maturation of 'Golden THB' papaya somatic embryos. The MM, supplemented with malt extract at 0.153 g L-1, promoted the greatest development of normal somatic embryos (18.28 SE calli-1), that is, two cotyledonary leaves produced 36.56 SE calli-1. The supplementation with 0.45 mM myo-inositol provided the highest germination percentage (47.42%) and conversion to emblings.

Evaluation of the micropropagation potential of curauá pineapple hybrids for fiber production

ABSTRACT Plant fiber is a renewable and biodegradable material that can be used effectively to reinforce various composites. Pineapple hybrids selected for their fiber quality are in the phase of agronomic validation in Brazil by the Embrapa Cassava and Fruits research unit. The selection of a hybrid for large-scale fiber production depends on obtaining a large number of seedlings. This study evaluated the morphogenetic response and propagation potential of eight hybrids of Ananas comosus var. erectifolius, for the purpose of producing high-quality seedlings on a large scale. Stem and crown buds were reduced and placed in MS nutritive medium supplemented with BAP at 0.5 mg L-1, NAA at 0.01 mg L-1 and Phytagel® at 2.5 g L-1. After 45 days, the number of oxidized, contaminated and surviving buds was determined. Swollen buds and plantlets were transferred to a multiplication medium containing MS sucrose, salts and vitamins. The propagation potential was evaluated based on the geometric growth rate among sub-cultures. The FIB-NEG hybrid presented the best results for the establishment phase (40.28%). The best propagative potential was obtained from crown buds with the highest values for FIB-EST (3.93), FIB-MIN (3.91) and FIB-BOY (3.91) hybrids.

In vitro conservation and regeneration of Laelia anceps Lindl

Laelia anceps Lindl is an orchid endemic to Mexico that is endangered of extinction as a result of the fragmentation of its habitat and the overexploitation of its natural populations. Therefore, programs aimed at the conservation and propagation of this genetic resource should be implemented. The objective of this study was to develop a protocol for in-vitro conservation and regeneration of L. anceps germoplasm. To establish the in-vitro conservation, this study different concentrations (¼, ½, and ¾) of MS salts (Murashige and Skoog), (0.5, 1, and 2 mg L−1) of two growth inhibitor abscisic acid (ABA) and of paclobutrazol (PBZ), and (10, 20, and 30 g L−1) the osmoregulator polyethylene glycol (PEG-8000) were evaluated. For in vitro regeneration, different concentrations (0, 0.5, 1, and 2 mg L−1) of Thidiazuron (TDZ) and (0, 0.5, 1, and 2 mg L−1) of 6-benzylaminopurine (BAP) were evaluated. 2 mg L−1 of PBZ reduced the development and growth in vitro of L. anceps, without drastically affecting the survival of the shoot. The higher number of shoot per explant (7.28) was achieved with the addition of 2 mg L−1 of BAP to the culture medium. Finally, a 90% survival was achieved during the acclimatization process. In conclusion, our results are a possible option in the establishment of strategies for in-vitro conservation and propagation of L. anceps, and is suitable for use in the preservation and propagation of other orchid species as part of species genetic improvement.

2- Isopentenyladenine in the induction of direct somatic embryogenesis capacity of Coffea arabica L.

ABSTRACT: This study evaluated the effect of different concentrations of 2-isopentenyladenine (2-iP) on the direct somatic embryogenesis capacity of the Mundo Novo cultivar of Coffea arabica. Leaf explants were cultivated with half the MS salt concentration and the addition of sucrose (20gL-1) and 2-iP (0; 2.5; 5; 7.5 and 10µM). The 2-iP doses of 7.5 and 10µM produced the greatest responses with respect to the percentage of explants with embryogenic structures and the size of the embryogenic structures. However, the greatest production of somatic embryos occurred on the explants treated with 10µM of 2-iP, followed by 7.5µM, whereas their production was absent or reduced with 0 and 5µM, respectively.

Elicitation of silver nanoparticles enhanced the secondary metabolites and pharmacological activities in cell suspension cultures of bitter gourd.

This study describes the influence of bio-synthesized silver nanoparticles (AgNPs) on phytochemicals and their pharmacological activities in the cell suspension cultures (CSC) of bitter gourd. To standardize the effect of sucrose, plant growth regulators, medium, AgNPs and growth kinetics for the biomass and bioactive compounds accumulation in CSC of bitter gourd. The medium comprising MS salts, sucrose (30g/L) with 2,4-D (1.0mg/L) and TDZ (0.1mg/L) at 28 days of CSC was appropriate for biomass and bioactive compound accumulation. The contents of silver, malondialdehyde and hydrogen peroxide were highly elevated in AgNPs (10mg/L)-elicited CSC when compared with non-elicited CSC. AgNPs (5mg/L) elicited CSC extracts had significantly enhanced the production of total phenolic (3.5 ± 0.2mg/g), and flavonoid (2.5 ± 0.06mg/g) contents than in the control CSC extracts (2.5 ± 0.1 and 1.6 ± 0.05mg/g). AgNPs (5mg/L) elicited CSC showed a higher amount of flavonols (1822.37µg/g), hydroxybenzoic (1713.40µg/g) and hydroxycinnamic (1080.10µg/g) acids than the control CSC (1199, 1394.42 and 944.52µg/g, respectively). Because of these metabolic changes, the pharmacological activities (antioxidant, antidiabetic, antibacterial, antifungal and anticancer) were high in the AgNPs (5mg/L)-elicited CSC extracts in bitter gourd. The study suggested the effectiveness of elicitation process in enhancing the accumulation of phenolic compounds and pharmacological activities. AgNPs-elicited CSC offered an effective and favorable in vitro method to improve the production of bioactive compounds for potential uses in pharmaceutical industries.

Biorreator na micropropagação de abacaxi ornamental

Ornamental pineapple is a hard plant with significant landscaping value. Typically, conventional propagation is performed by clump division with low yields, and may even spread diseases. Plant tissue culture is viable, yielding plants with a high phytosanitary and genetic quality over a short time period. This study aimed to verify the in vitro multiplication of ornamental pineapple plants (Ananas comosus var. bracteatus L.) in different micropropagation systems, in association with BAP concentrations. Plants with about 2 cm were used, transplanted to the different treatments: bioreactor, natural ventilation and conventional micropropagation, combined with 3 BAP concentrations (0, 1 and 2 mg L-1). The basic medium used consisted of MS salts. The highest number of shoots and in vitro culture growth were obtained with the use of bioreactor and culture medium containing 2 mg L-1 BAP. The temporary immersion bioreactor allows air renewal inside the bottles, leading to a better performance of in vitro cultivation of ornamental pineapple, when compared to conventional micropropagation.

Establecimiento in vitro de dos cultivares liberados de frutillas: fresa y frambuesa

El desarrollo de protocolos para la producción de plantas sanas in vitro es importante para satisfacer la demanda creciente de frutillas en el norte de México como una alternativa a los frutales tradicionales como nogal y manzano. Se evaluaron técnicas de establecimiento in vitro de fresa y frambuesa, a partir de explantes meristematicos, ápices y entrenudos. Se utilizó un método de desinfección convemcional, donde se utilizaron agentes desinfectantes como solución etanólica al 70%, luego se sumergieron en solución solución comercial al 3% de hipoclorito de sodio: agua (1:6), durante 15 min. Por último, en solución de timerosal® al 0.05% por 15 min. Se evaluaron ápices en el caso de fresa ápices, y de meristemos y entrenudos en el de frambuesa. Para el establecimiento se utilizaron sales Murashige y Skoog adicionados con citocinina 6-bencilaminopurina (BAP), ácido giberélico (GA) e ácido indolbutírico (AIB) en diferentes concentraciones para el caso de fresa. En frambuesa, se utilizaron sales de MS adicionadas con BAP, tidiazuron (TDZ), GA y AIB. Se evaluaron en ambos, los porcentajes de establecimiento, necrosis y contaminación. La siembra de ápices en el caso de fresa y su sistema de desinfección, permitió su establecimiento in vitro, las variables se analizaron mediante pruebas no paramétricas mostraron que el mejor tratamiento, fue TT1 con 70% de efectividad. Para frambuesa, el caso de entrenudos fue mejor durante el establecimiento que en el caso de meristemos, y el mejor tratamiento fue el TT4, con BAP + GA sin auxinas, con 57% de efectividad.

Development of simple in-vitro protocol for screening low soil nitrogen-efficient maize lines

In order to fast track and compliment conventional maize breeding programme designed for low nitrogen (N) environments, seeds of eight low soil N-tolerant maize lines were sown in the field under no nitrogen and 40 kgN/ha for in vivo evaluation with a view to develop simple protocols for screening maize lines for low nitrogen-efficient through tissue culture technique. Maize plants used for tissue culture (in vitro) were selfed, cobs were harvested fourteen days after pollination before the kernels were disinfected. The immature embryos were excised and cultured on modified Murashige and Skoog basal medium (MS) containing 100% Nitrogen (1650 mg/l), 50% Nitrogen (825 mg/l), 25% Nitrogen (412.5 mg/l), 0% Nitrogen of normal ammonium nitrate in MS salt formulation. Results obtained revealed three lines (LNTP-YC6, TZPB Prol C3 and SW5-OB-BPR-1) had higher grain yield than other maize lines under no fertilizer (0 kgN/ha) and 40 kgN/ha fertilizer applications (P = 0.01) while two lines (TZPB Prol C3 and LAPOSTA SEQUIA C6) performed better than others with or without ammonium nitrate under in-vitro condition (P = 0.01). TZPB Prol C3 was consistent in both in-vitro and in-vivo conditions. Also, MS medium without ammonium nitrate appeared to be more suitable for screening for low N-tolerant maize genotypes.

Cryotherapy by encapsulation-dehydration is effective for in vitro eradication of latent viruses from ‘Marubakaido’ apple rootstock

Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) are several major viral pathogens of apple trees, responsible for substantial damage to the world’s apple industry. This study aimed to evaluate the effectiveness of the encapsulation-dehydration cryopreservation technique to eradicate these viral pathogens from in vitro shoot tips excised from ‘Marubakaido’ apple rootstock cultures. Axillary shoot tips were excised from in vitro cultures, encapsulated in alginate beads, precultured in MS salts, dehydrated in a laminar flow hood, immersed in liquid nitrogen, then warmed and recovered on medium. After LN exposure, in vitro rooting and acclimatization, recovered ‘Marubakaido’ plants exhibited 52% survival and 35% regrowth without callus formation. After 8 months of regrowth, PCR analyses revealed that all the plants were free of ACLSV and ASPV, but 2 out of 20 recovered plants were still infected with ASGV. This is the first report in Brazil of the application of cryotherapy to eradicate viral complexes in Malus. Cryotherapy can facilitate the production of virus-free plants by producing high quality plant material.

Determination of Optimal Hormone and Mineral Salts Levels in Tissue Culture Media for Callus Induction and Growth of Industrial Hemp (<i>Cannabis sativa</i> L.)

Historically, industrial hemp (Cannabis sativa L.) has been a valuable source of metabolites and compounds, such as cannabidiols. There is a need for large amounts of plant tissue to be grown under controlled environments, and plant tissue culture is one unique way to yield this tissue. The purposes of this study were to determine: 1) the optimal concentrations (μM)/ratios of auxin:cytokinin in media and; 2) the optimal mineral salts formulation for callus induction and callus growth in select hemp cultivars. To find the optimal concentration/ratios, 16 different combinations of auxin:cytokinin and three different mineral salts formulations were evaluated. The three mineral salts formulations tested were MS salts, MB5D1K and an MTSU formulation. The top performing hormone formulations were determined to be equal concentrations (1:1, 2:2, 3:3 μM) of auxin and cytokinin. The top performing media hormone formulations for callus induction were determined to be 2:1, 2:2, 2:3, and 3:2 μM (auxin:cytokinin). The optimal mineral salts formulation was determined to be MD5D1K. Therefore, the overall optimal media formulation for hemp callus production would be MB5D1K salts with the concentration/ratio of 2:2 μM (auxin:cytokinin).

An efficient in vitro propagation protocol of Dianthus giganteiformis Borbas subsp. kladovanus (Degen) Soo

Dianthus giganteiformis subsp. kladovanus is an endemic, endangered, horticulturally appealing perennial plant that can be used for the revegetation of sand dunes of the Danube region. The appropriate method for its effective production is micropropagation. For this reason, the experiments were conducted in order to establish an efficient protocol for the micropropagation of this subspecies. The sterile culture was initiated from seeds collected in situ and the germination percentage was high (88%). According to the results obtained in this study, the multiplication phase should be performed on an MS medium enriched with 0.1 mg/L BAP and 0.1 mg/L NAA. The concentration of MS salts significantly influenced rooting, and higher rooting percentages were obtained on reduced MS media (91.7 - 95%) than on MS media (73.4 - 76.7%). The addition of NAA slightly increased rooting percentage (up to 95%). Obtained microplants were successfully acclimatized (83.3%) in a substrate composed of peat and sand (1: 1; v/v). Using the protocol presented in this paper, the efficient propagation of D. giganteiformis spp. kladovanus can be achieved for rapid plant production aimed at revegetation, biodiversity protection and floricultural production of this species.

Micropropagation response of three grape vines (Vitis vinifera L.) cultivars to ferrous, nitrate and growth regulators

Three Grapevine (Vitis vinifera L.) cultivars, Kamaly, Taify and Rash Mew were propagated by tissue culture technique and a reproducible and reliable protocol was developed through this investigation. Single nodes of grapevine were initiated on MS medium after getting healthy aseptic cultures by their surface sterilization with 0.15% HgCl2 for 6 minutes. The shoots were multiplied by the addition of BA, Fe and N. The best multiplication parameters were obtained from the Kamaly cultivar grown on MS medium supplemented with 3.0 mgl-1 BA and 10 mgl−1 Fe. Whereas, the culture of Taify grape cultivar on MS medium supplemented with 10 mgl−1 N produced the longest shoots (3.00 cm). The best rooting results were obtained for Taify and Rash Mew cultivars especially when grown on full MS salts strength media supplemented with 1.0 mgl−1 NAA. A high survival rate of gradually acclimatized Rash Mew plantlets was obtained (100%). The best culture mixture for acclimatization stage was composed of peatmoss and perlite.

Improved production of Oroxylum indicum (L.) Kurz by altering the salts of the medium

The present study was conducted to test the effects of KNO3, KH2PO4, and CaCl2 on shoot multiplication, root proliferation, and accumulation of phytochemicals in in vitro cultures of Oroxylum indicum. The results indicate that modifying the MS salt formulation in relation to particular inorganic nutrients highly affected shoot multiplication, root proliferation, and accumulation of flavonoids in in vitro cultures. A concentration of 0.60 g L−1 CaCl2 resulted in the highest frequency of shoot regeneration (5.6 shoots per explant). A concentration of 0.40 g L−1 CaCl2 resulted in the highest frequency of root regeneration (7.8 roots per shoot). Modifications of the concentrations of inorganic salts were also found to be advantageous for production media for both multiple shoots and shoot-derived root in vitro cultures. Multiple shoots generated on shoot induction medium with a concentration of 0.60 g L−1 CaCl2 and roots generated on root induction medium with a concentration of 1.5 g L−1 KNO3 yielded about a five times higher flavonoid level than cultures generated on control medium respectively.

PROPAGAÇÃO IN VITRO DE <i>Casearia sylvestris</i> SWARTZ (SALICACEAE)

Com o objetivo obter a propagação in vitro de Casearia sylvestris, árvore nativa das florestas brasileiras, estudos de germinação in vitro das sementes foram realizados e as plântulas assépticas obtidas foram usadas como doadoras de explantes na micropropagação. Sementes maduras e imaturas, armazenadas por 10 dias a 10 ou 25°C foram inoculadas em meio contendo 10% de sais de MS e mantidas na luz (fotoperíodo de 16 horas) ou escuro contínuo, a 25°C. Para sementes imaturas, testou-se ou não ácido giberélico (GA3) na concentração de 1,5 mg l-1. Das plântulas obtidas, segmentos cotiledonares e apicais foram retirados e cultivados em meio MS completo com cinco combinações de 6-benzilaminopurina (BAP) e ácido naftalenoacético (ANA): 0,0; 0,25; 0,5; 1,0; 2,0 e 0,0; 0025; 0,05; 0,1; 0,2 mg L-1, respectivamente. As brotações aéreas formadas in vitro foram cultivadas em meio MS contendo 0,0 ou 1,5 mg L-1 de ácido indol-3-butírico (IBA) para o enraizamento. O maior percentual de germinação (60%) ocorreu em sementes maduras expostas à luz, independentemente dos demais tratamentos, podendo ser consideradas fotoblásticas positivas. A maior percentagem de germinação das sementes imaturas (27%) ocorreu quando previamente armazenadas a 10°C, com ou sem GA3. A indução de brotos aéreos foi mais eficiente em segmentos apicais e em meio contendo a menor combinação de BAP e ANA ou isento destes reguladores de crescimento. O maior número de raízes ocorreu em brotos obtidos a partir de segmentos cotiledonares, com ou sem AIB. Após a aclimatização, 83% das plantas sobreviveram.

Improving In Vitro Somatic Embryos Production of Medjool and Khalas Date Palm Cultivars via Modification of Ammonium and Potassium Nitrate

Date palm is one of the most economically powerful fruit tree grown in Egypt. Although Egypt tops the list of date's product in the world. Egyptian date palm industry suffers from many constraints. Low-quality varieties and limited conventional propagation methods are the main obstacles. Extension of date palm plantations in various areas of Egypt and replanting trees to compensate loss due to diseases or human factors are reduced expected to lack adequate planting materials. The knowledge of the interplay between some fundamental medium parameters allows not only the optimization of the micropropagation system but also gives models to investigate and rationalize the process of induction and development of somatic embryogenesis itself. In vitro growth and development of produced embryos and plantlets has been improved by using of full MS salt strength. MS medium modification of some nutrients concentrations, i.e. NH4NO3(1237.5 mg/l) which recorded (13.20 and 9.60 embryos/explant for Medjool and Khalas respectively) at the end of three subculture. KNO3 which gave a higher score of mature somatic embryos (14.40 and 10.00 embryo/culture for Medjool and Khalas respectively) was obtained with 1425 mg/l KNO3 after three subcultures as compared to those obtained from any other treatment combination. The morphological response of somatic embryos production is controlled by some internal factors that fall under the influence of the genetic make-up of the plant and specialized for each genotype that is responsible for the production of somatic embryos (Medjool and Khalas cultivars). The genetic make-up is a decisive factor during somatic embryos production. There are differences between the two cultivars named, Medjool and Khalas. The produced somatic embryos at germination stage were transferred from the two experiments namely ammonium nitrate and potassium nitrate to multiplication and rooting stages for more in vitro growth for 12 weeks at three re-cultures. Individual shoots were cultured on modified MS basal medium in addition to IBA (1.0 mg/l), sucrose (30 g/l) and solidified with phyto-agar (8.0 g/l) for more in vitro growth and development. In vitro plantlets were transferred to acclimatization stage in plastic pots 5*18 cm diameter containing peatmoss, perlite and washed sand at equal volume. In vitro date palm plantlets produced from rooting stage grow well in the greenhouse during acclimatization stage without morphological abnormality. The growth and development of Medjool cultivar were better than Khalas during in vitro culture and ex vitro acclimatization.