Encapsulation of nodal segments of Cassia angustifolia Vahl. for short-term storage and germplasm exchange

The present study described the encapsulation of nodal segments of Cassia angustifolia Vahl. excised from 1-month-old in vitro raised cultures for short-term conservation and propagation. Various concentrations and combinations of gelling matrix (sodium alginate) and complexing agents (calcium chloride) were tested to prepare uniform beads. The ideal beads were obtained through a combination of 3 % sodium alginate and 100 mM calcium chloride. The maximum conversion response (94 %) of encapsulated beads was obtained in Murashige and Skoog’s medium (MS medium) supplemented with 2.5 μM benzyladenine (BA) and 0.4 μM α-naphthalene acetic acid (NAA) after 6 weeks of culture. The encapsulated and non-encapsulated nodal segments were also stored at 4 °C for different time periods (0, 1, 2, 4, 6 and 8 weeks). The regenerated microshoots were best rooted in optimized rooting medium that comprised half-strength MS + 1.0 μM indole-3-butyric acid (IBA) + 5.0 μM phloroglucinol (PG) for the production of complete plantlets. The regenerated plantlets were successfully hardened and acclimatized in natural conditions with 70 % survival rate.     

Encapsulation of nodal cuttings and shoot tips for storage and exchange of cassava germplasm

We report the encapsulation of in vitro-derived nodal cuttings or shoot tips of cassava in 3% calcium alginate for storage and germplasm exchange purposes. Shoot regrowth was not significantly affected by the concentration of sucrose in the alginate matrix while root formation was. In contrast, increasing the sucrose concentration in the calcium chloride polymerisation medium significantly reduced regrowth from encapsulated nodal cuttings of accession TME 60444. Supplementing the alginate matrix with increased concentrations of 6-benzylaminopurine and alpha-naphthaleneacetic acid enhanced complete plant regrowth within 2 weeks. Furthermore, plant regrowth by encapsulated nodal cuttings and shoot tips was significantly affected by the duration of the storage period as shoot recovery decreased from almost 100% to 73.3% for encapsulated nodal cuttings and 94.4% to 60% for shoot tips after 28 days of storage. The high frequency of plant regrowth from alginate-coated micropropagules coupled with high viability percentage after 28 days of storage is highly encouraging for the exchange of cassava genetic resources. Such encapsulated micropropagules could be used as an alternative to synthetic seeds derived from somatic embryos.     

Nutrient-encapsulation of potato nodal segments for germplasm exchange and distribution

Nutrient-encapsulation technique using in vitro grown nodal segments was developed as an alternative method for distribution of potato germplasm. The nodal cuttings of two potato genotypes, Kufri Jyoti and Kufri Lauvkar, were encapsulated in calcium-free Murashige and Skoog's (MS) medium containing either 2 or 3 % sodium alginate and 0, 1, 2 or 3 % saccharose. The encapsulated segments were stored in tubes with or without semisolid MS medium, and incubated in the dark at 25 ± °C for 3 to 6 weeks. Presence of saccharose in the beads was found detrimental for regrowth of new shoots. In absence of saccharose, about 82 % and 53 % encapsulated segments initiated regrowth after 3 and 6 weeks of dark storage, respectively, in tubes containing MS medium. Storage in empty tubes deteriorated the encapsulated segments, and depressed shoot formation. For potato germplasm distribution, the encapsulated segments can be transported in small tubes containing semisolid MS medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nutrient alginate encapsulation of nodal segments of Althaea officinalis L., for short-term conservation and germplasm exchange

Abstract

In the present study, an alternate method for germplasm storage in the form of artificial seeds was standardized via nodal explants excised from in vitro proliferated shoots. The explants were encapsulated using sodium alginate and calcium chloride as gelling matrix. For development of root along with shoot, excised nodal segments were pretreated with ½ MS medium along with 20 μM IBA for 24 h and encapsulation was carried thereafter. Combination of 3% sodium alginate augmented with 100 mM CaCl₂.2H₂O was found appropriate for the formation of clear and uniform beads and subsequent conversion of encapsulated nodal segments into plantlets. Maximum (66%) encapsulated nodal segments were converted into plantlets on MS medium supplemented with 7.5 μM BA and 0.5 μM NAA after eight weeks. Regeneration frequency of auxin-pretreated encapsulated and non-encapsulated nodal segments (stored at 4 ºC) was evaluated at different storage time (0 to 6 weeks). After four weeks of storage, encapsulated propagules exhibited highest conversion response on the optimized medium after eight weeks of culture. Plantlets were hardened and established with success in ex vitro conditions. Conversion of synthetic seeds into plantlets was observed when these were directly sown in autoclaved Soilrite^TM (Keltech Energies, Bangalore, India).     

Encapsulation, cold storage, and growth of Hibiscus moscheutos nodal segments

Nodal segments of Hibiscus moscheutos (hardy hibiscus) were excised from proliferating axillary shoot cultures and encapsulated in high density sodium alginate hardened by 50 mM CaCl₂. Nodal segments 4 mm long grew as well as and were easier to encapsulate than 8 mm long nodal segments. Although nodal segments grew regardless of the concentration of sodium alginate, 2.75% was determined to produce the highest quality encapsulated nodal segments beads (sufficient alginate coating and ease of use) because of the viscosity produced by the 2.75% sodium alginate solution. When encapsulated segments were stored at 5°C they did not grow in light or darkness. During the first month on fresh proliferation medium under normal incubation conditions following 5°C storage in the dark for up to 24 weeks, root number and root and shoot elongation were inhibited linearly as storage time increased. All encapsulated nodal segments survived 24 weeks of 5°C storage in two separate experiments. In fact, 80% of encapsulated hardy hibiscus nodal segments survived refrigerated storage for 1½ years (78 weeks) and after 3 months on proliferation medium, the nodal segments produced nearly the same length axillary shoots with the same number of axillary nodes per shoot as compared to encapsulated segments either not stored at 5°C or stored for 24 weeks at 5°C. Growth from encapsulated and cold-stored ‘Lord Baltimore’ nodal segments was more vigorous than from ‘Southern Belle’ nodal segments.     

Alginate-encapsulation of nodal segments for propagation, short-term conservation and germplasm exchange and distribution of Eclipta alba (L.)

Nodal segments obtained from in vitro proliferated shoots of Eclipta alba (L.) Hassk, were encapsulated in calcium alginate beads for large-scale clonal propagation, short-term conservation and germplasm exchange and distribution. The best gel complexation was achieved using 3% sodium alginate and 100 mM CaCl₂·2H₂O. Maximum percent response (100%) for conversion of encapsulated nodal segments into plantlets was obtained on 0.7% agar-solidified full-strength MS medium containing 0.88 μM BAP. Encapsulated nodal segments could be stored at low temperature (4°C) up to 60 days with a survival frequency of 51.2%. The well-developed plantlets regenerated from encapsulated nodal segments were hardened-off successfully with 90% survival frequency.     

Histochemistry and Senescence of Colleters of Allamanda cathartica (Apocynaceae)

Allamanda have colleters on the adaxial bases of petiole, bractsand bracteoles. Each colleter is finger shaped, differentiatedinto a long head on a short stalk. Vasculature to the colleteris absent but branched laticifers are present among the centralcells. Secretion of the colleter contains glucose and rhamnose.The major elements present in the exudate are Na, Fe and Zn.Starch, protein and lipids were identified, with lipids themajor component. Petiolar colleters are persistent and becomestiff due to the lignification occurring in the walls of epithelialand central cells. Because of over-lignification in the centralcells, the cell lumen is highly reduced. Allamanda, colleter, laticifer, secretion, senescence     

Encapsulation technology for short-term storage and conservation of a woody climber, Decalepis hamiltonii Wight and Arn.

An efficient protocol was developed for short-term storage and conservation of a woody medicinal climber, Decalepis hamiltonii, using encapsulated nodal segments. The encapsulation of nodal segments was significantly affected by the concentrations of sodium alginate (Na-alginate) and calcium chloride (CaCl₂·2H₂O). A gelling matrix of 4?% Na-alginate and 100?mM CaCl₂·2H₂O was found most suitable for the production of ideal Ca-alginate beads. Maximum shoot re-growth (77.00?±?2.09?%) was recorded on Murashige and Skoog (MS) basal medium supplemented with 5.0???M 6-benzyladenine (BA), 0.5???M indole-3-acetic acid (IAA) and 30.0???M adenine-sulphate (ADS). Microshoots, recovered from encapsulated nodal segments (capsule) were best rooted on half-strength MS medium containing 2.5???M ??-naphthalene acetic acid (NAA). Complete plantlets (with shoot and root) were successfully acclimatized and established in field where they grew well without any detectable variation.     

Micropropagation of Coleus blumei from nodal segments and shoot tips

A rapid and highly-effective method for micropropagation from nodal segment and shoot tip explants was established for Coleus blumei Benth. Nodal segments and shoot tips were inoculated on MS medium containing 0.7 % agar, 3 % commercial sugar, and different combinations of 6-benzyladenine (BA) with indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Hundred percent shoot induction from both explants was achieved on the medium containing BA (2 mg dm⁻³) and NAA (1 mg dm⁻³). Shoot tips were proved to be the better explant in comparison to nodal segments in having high rate of shoot induction and more number of shoots. The same media conditions were found suitable for shoot multiplication. Multiplied shoots rooted best on MS medium supplemented with IBA (2 mg dm⁻³). Micropropagated plants were successfully established in soil after hardening, with 100 % survival rate.     

Greenhouse growth and acclimatization of encapsulated Hibiscus moscheutos nodal segments

Leafless nodal segments (4 ± 1 mm long) of hardy hibiscus were excised from in vitro proliferating microshoots, encapsulated in sodium alginate solidified with 50 μM CaCl₂, stored under refrigeration for 4 weeks in darkness, and then planted in the greenhouse. Planting in vermiculite and placing under intermittent mist was the best environment tested. If the encapsulated nodal segments were exposed to light for at least 2 weeks while in vitro in the laboratory prior to planting in the greenhouse, all survived, rooted, and produced shoots in the greenhouse. Rooting into the vermiculite was best if the encapsulated nodal segments were planted 1 cm deep and not covered. Anatomically, the new leaves that were produced from shoots that grew under mist in the greenhouse from encapsulated nodal segments were about the same thickness as leaves produced in vitro; had fewer intercellular spaces than the in vitro produced leaves; had palisade cells intermediate in length, and were intermediate for epicuticular wax formation between in vitro produced leaves and leaves on macrocuttings rooted in the greenhouse. The stomates on greenhouse shoots from encapsulated nodal segments closed similar to stomates on leaves on rooted macrocuttings, and were unlike in vitro produced leaves where the stomates remained open even when stressed. Storing and planting encapsulated nodal segments could allow producers to generate sufficient numbers of nodal segments, refrigerate them until needed, and facilitate greenhouse acclimatization and production of plants.     

Cryopreservation and long-term storage of pear germplasm

Summary Germplasm collections of vegetatively propagated crops are usually maintained as plants in fields or potted in greenhouses or screened enclosures. Safety duplication of these collections, as duplicate plants or separate collections, is costly and requires large amounts of space. Cryopreservation techniques which were recently developed for long-term storage of pear germalasm may offer an efficient alternative to conventional germplasm collection maintenance. Pear (Pyrus L.) germplasm may now be stored as seeds (species), dormant buds or pollen from field-grown trees, or shoot tips fromin vitro-grown plants (cultivars). Pear germplasm may now be cryopreserved and stored for long periods (> 100 yr) utilizing slow-freezing or vitrification ofin vitro-grown shoot-tips. Dormant bud freezing, pollen, and seed cryopreservation of other lines are being developed to complete the base collection forPyrus. This cryopreserved collection provides base (long-term) storage for the field-grown pear germplasm collection at the National Clonal Germplasm Repository, Corvallis, Oregon. Based on a presentation at the 1997 Congress on In Vitro Biology held in Washington, D.C., June 14–18, 1997.     

Encapsulation technology for short-term preservation and germplasm distribution of the African mahogany <Emphasis Type="Italic">Khaya senegalensis</Emphasis>

A protocol was developed for short-term preservation and distribution of the medicinal and timber plantation tree, Khaya senegalensis, using alginate-encapsulated shoot tips. The study assessed the effects of culture medium, storage temperature, auxin concentration and planting substrate on shoot regrowth or conversion into plantlets of four different clones. Optimal shoot growth was obtained, with high frequencies (92–100%) of shoot emergence, on Murashige and Skoog (MS) culture media containing 4.4 μM benzyladenine (BA). Encapsulated shoot tips survived longer at 25°C than at 4°C, with viability of 73–88% after 8 weeks. Conversion into plantlets was achieved on half-strength MS medium by pre-culture treatment of shoot tips with 49–490 μM indole-3-butyric acid (IBA) before encapsulation. Treatment with 245 μM IBA provided 52–98% conversion, and 90–95% of plantlets survived acclimatisation under nursery conditions. To eliminate the in vitro culture step after encapsulation, synthetic seeds were allowed to pre-convert before sowing directly onto a range of ex vitro non-sterile substrates. Highest frequencies of plantlet formation from pre-converted synthetic seeds (42–86%) were obtained by transferring synthetic seeds to organic compost, and these plantlets exhibited almost 100% survival in the nursery without mist irrigation. Pre-conversion is a novel and convenient method for producing synthetic seeds that are suitable for distribution to commercial nurseries.     

In vitro micropropagation from nodal segments of Cleistanthus collinus

Cleistanthus collinus Benth. was micropropagated using nodal explants on MS medium supplemented with 2.2 M benzyladenine (BA). April to June was the best time for initiating shoot cultures. Shoot proliferation was enhanced when the BA concentration was lowered to 1.1 M. Rooting was achieved on half-strenth MS medium with 22.8 M indole-3-acetic acid for 7 days and continuous darkness for the first 72 h of the 7 days.Abbreviations MS Murashige & Skoog's medium - WPM Woody Plant Medium - BA 6-benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthalenacetic acid     

硬枝黄蝉染色体计数与核型分析

以硬枝黄蝉Allamanda neriifolia幼胚为试验材料,对其体细胞染色体进行计数与核型分析。结果表明,硬枝黄蝉幼胚细胞含9对染色体,由中部或近中部着丝粒染色体构成。核型公式为2n=2x=6sm+12m。核型不对称系数为58.95%,核型分类属于2A型。     

Experimental microbial contamination and disinfection of dry (vapour) shipper dewars designed for short-term storage and transportation of cryopreserved germplasm and other biological specimens

Cryopreservation, storage and transport of cryopreserved germplasm without the risk of disease transmission is of great concern to animal and human health authorities. Here we report on the efficacy of microbial decontamination of the liquid nitrogen (LN) dry (vapour) shippers used for short-term storage and transportation of germplasm and other biological specimens. Dry shippers containing either a hydrophobic or a non-hydrophobic LN absorbent were experimentally contaminated with high titers of cultures of Pseudomonas aeruginosa, Escherichia coli, Staphylococus aureus, bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BHV-1). Biocidals with broad spectrum antimicrobial activity and gas vapours of formalin and ethylene oxide were used for disinfection of the dewars. Among the biocidals used, treatment with sodium hypochlorite solution, the quaternary ammonium-based disinfectants and peracetic acid were the most effective and useful for dry shippers with a hydrophobic LN absorbent. None of the bacterial or viral microorganisms were detected in samples of semen and embryos stored in dry shippers following their disinfection with these biocides. An application of some other disinfectants, due to their foaming properties or to the permeability of the absorbent hydrophobic membrane (HM) was not effective or may have caused irreversible damage to the LN absorbent. Gas sterilization by ethylene oxide in contrast to formalin was fully effective for both types of dry shippers.     

Bud emergence and shoot growth from mature citrus nodal stem segments

Bud emergence and shoot growth from adult phase citrus nodal cultures were studied using Citrus mitis (calamondin), Citrus paradisi (grapefruit), and Citrus sinensis (sweet orange). The effects of 6-benzyladenine (BA), indole 3-acetic acid (IAA), and citrus type on shoot quality and growth of mature bud explants from greenhouse grown trees were determined using a 2-component mixture-amount × citrus type experiment. BA increased shoot number and IAA improved shoot growth. The best shoot quality (fewer shoots but large shoots) was obtained with 1 μM IAA for calamondin, 15.5 μM IAA for sweet orange, and 30 μM IAA for grapefruit. Grapefruit exhibited substantial leaf abscission compared to calamondin and sweet orange. Four factors (AgNO₃, silver thiosulphate (STS), CaNO₃, or gelling) were screened individually for their efficacy in reducing leaf abscission. Five factors (AgNO₃, gelling, MS ion concentration, plant growth regulator and venting) were investigated to identify potential combinations for reducing leaf abscission and maximizing shoot growth and bud emergence. The factor combination identified as most effective in minimizing leaf drop, promoting shoot growth, and maximizing bud emergence for grapefruit was 2 mg l^?1 AgNO_3, Gelrite, 1 × MS ion concentration, 30 μM IAA, and vented.     

Organogenesis and efficient in vitro plantlet regeneration from nodal segments of <Emphasis Type="Italic">Allamanda cathartica</Emphasis> L. using TDZ and ultrasound assisted extraction of quercetin

The present investigation was carried out to evaluate the instigative effect of thidiazuron (TDZ) on multiple shoot induction from nodal segments of Allamanda cathartica and estimated the flavonoid yield among the regenerants. High rate of shoot bud induction was achieved on Murashige and Skoog (MS) medium augmented with 0.3 µM TDZ from nodal segments exposed for 30 days. However, for shoot proliferation and elongation, TDZ exposed cultures were further cultured on MS medium devoid of TDZ and/or supplemented with different concentration of 6-benzyladenine (BA) and Kinetin (Kn). BA at 2.5 µM gave the maximum mean number of shoots (44.00?±?1.30) and shoot length (7.50?±?0.21 cm) per explant after 12 weeks of incubation in the secondary medium. The response of explant was influenced by the collection time. The highest rooting in the microshoots (5 cm) was achieved on 1/2 MS liquid medium supplemented with 0.5 µM Indole-3 butyric acid (IBA) which produced 4.50?±?0.16 mean roots/shoot with 4.05?±?0.17 cm mean root length. The leaves of 30 day old acclimatized plantlets were used for phytochemical screening. Ultrasonication mediated extraction and quantification of bioactive flavonoid namely quercetin through colorimetry and mass spectrometry analysis from the leaves of regenerants. Extraction was processed in methanol using 2 g leaf sample through sonication. Total yield of flavonoids and quercetin content was found to be maximum in 2.5 µM BA treated plants with respect to control and other treated samples. The concentration of total flavonoids was estimated to be 172.90 mg QE/g which yielded 51.39 mg/g quercetin. The study ensures a rapid cultivation of plantlets, thus enhancing the biomass production which may be utilized in the isolation and quantification of other biological potential compound for the use in treatment of various ailments.     

桃花粉低温和超低温保存方法比较研究

桃(Prunus persica(L.)Batsch)是我国重要的无性繁殖作物种质资源,目前主要保存于3个国家无性繁殖作物种质圃。随着以茎尖、花粉、休眠芽为保存载体的超低温保存技术的发展,超低温保存已成为无性繁殖作物重要备份保存方式。本研究以15份桃种质花粉为研究对象,开展含水量、回湿处理和保存温度(4℃低温保存和液氮超低温保存)对保存后花粉离体萌发率的影响研究。研究结果:明确了桃种质花粉超低温保存的含水量;揭示了回湿处理对部分桃种质花粉超低温保存产生显著影响;超低温保存后花粉离体萌发率最高可达83%;4℃低温保存和超低温保存比较研究结果表明,超低温保存4年后14份桃种质花粉离体萌发率仍可保持30%以上,11份桃种质花粉离体萌发率与保存前花粉离体萌发率相比无显著变化甚至显著提高,而4℃低温保存的花粉离体萌发率降至0。该研究为国家种质库建立花粉规模化超低温保存提供技术支撑。