Recurrent production of plants of black plum, Syzygium cuminii (L.) Skeels, a myrtaceous fruit tree, from in vitro cultured seedling explants

 The epicotyl segments bearing scaly leave(s), excised from in vitro grown seedlings of Syzygium cuminii, produced multiple shoots when cultivated on Murashige and Skoog's (MS, 1962) medium supplemented with different concentrations of BA (0–2 mg l^–1). The optimum response was recorded on the medium containing 1 mg l^–1 BA. An average of 8.6 shoots per explant were produced 60 days after inoculation, following transfer to fresh medium after 30 days. The shoots were excised, and the residual explants were transferred to fresh medium, where they again developed shoots. Up to five such passages resulted in the production of shoots from the repeatedly subcultured original explants. However, during the fifth passage, organogenic response was negligible and the explants turned brown thereafter. Following repeated harvesting of shoots and subculture of the residual explants, an average of 29 shoots per explant was obtained. The in vitro developed shoots produced roots when transferred to Knop's medium supplemented with 2% sucrose and 1 mg l^–1 IAA. The developed plantlets were planted in soil and transferred to fields after an acclimatization period of 7–8 months. These plants have been thriving well for more than 3 years. The nodal explants excised from in vitro developed shoots and plants also exhibited a similar response when cultured on MS+1 mg l^–1 BA. Thus, a protocol has been developed to raise plants of S. cuminii at any time of the year. Received: 1 December 1998 / Revision received: 1 July 1999 · Accepted: 12 July 1999     

In vitro reinvigoration of mature olive trees (Olea europaea L.) through micrografting

Summary Portions (1.0–1.5 cm long) of terminal shoots from selected mature treesOlea europaea L. cv. Arbequina, micrografted in one phase ontoin vitro juvenile shoots, resulted in the restoration of shoot-bud proliferation and rooting competence. Although higherin vitro survival rates were obtained after a second repeated micrografting, the reinvigoration ratio of the regenerated shoots, indicated by proliferation and rooting ability, was not improved after two phases of micrografting. Thus, one-phase micrograft allows for a successful micropropagation system for olive trees. The cuttings obtained from successive pruning of plants produced through micrografting and growth in soil showed complete restoration of rooting competence, with rooting percentages similar to those of juvenile microshoots.     

Rapid clonal propagation of guava through in vitro shoot proliferation on nodal explants of mature trees

In vitro clonal propagation of guava Banaras local was achieved by culturing nodal explants of mature trees on Murashige and Skoog (MS) revised medium supplemented with 4.5 M 6-benzyladanine (BA) alone or in combination with either 0.6 M indole-3-acetic acid (IAA), 0.5 M indole-3-butyric acid (IBA) or 0.3 M gibberellic acid (GA₃). Multiple shoots were induced to form by enhancement of axillary branching and BA (4.5 M) without any auxin and gibberellin was found to give best shoot multiplication rate. In this medium 3–6 shoots developed on explants collected from field-grown plants and 5–10 shoots developed on explants taken from in vitro proliferated shoots within 12 wk of culture. A prior transfer of shoot clumps to a medium containing a lower concentration of BA (0.5 M) before harvesting of cuttings for rooting allowed rapid extension growth and increased the number of usable shoots per culture. Adventitious rooting occurred after subculturing excised shoots on a medium containing 1/2 strength MS salts, 1.5% sucrose, 1 M each of IBA and -naphtha-leneacetic acid (NAA), and 1 gl^-1 activated charcoal. Regenerated plantlets were successfully established on soil.     

Micropropagation ofDalbergia sissoo from nodal explants of mature trees

A method for micropropagation ofDalbergia sissoo has been developed. Single node segments obtained from coppice shoots of a mature tree (20 – 25 year old) produced 3–4 shoots per explant on Murashige and Skoog (MS) medium containing 4.4 x 10⁻⁶ M benzylaminopurine (BAP) and 4.4 × 10⁻⁷ M of Β-naphthoxy acetic acid (NOA) (shoot multiplication medium) within 4 weeks. Thein vitro regenerated shoots were 3 – 4 cm in length and provided 2 to 3 culturable nodal segments which on shoot multiplication medium again produced 3–4 shoots. Following this procedure 18–24 shoots were produced from single nodal segment within 60 d. 80 % of the shoots directly produced five roots when they were firstly treated with MS medium supplemented with 10⁻⁵ M indole-3-butyric acid (IBA) and subsequently transferred to half strength liquid MS medium containing 1 % activated charcoal followed by half strength liquid MS free hormones, vitamins and activated charcoal. Thein vitro raised plants were hardened for survival after transplantation to soil by exposing them to various humidity conditions, gradually from higher to low, with nearly 100 % transplant success. Acknowledgement: Authors are grateful to CSIR and DST, New Delhi for financial assistance.     

Micropropagation of Tamarix gallica from nodal explants of mature trees

Tamarix gallica L. was micropropagated from four-to six-node explants taken from mature trees. Shoot proliferation was induced on Linsmaier and Skoog medium containing 30 g l^-1 sucrose, 7 g l^-1 agar, 200 mg l^-1 reduced glutathione (basal medium) and supplemented with 3.3 M benzyladenine. Adding 0.5 or 1.0 M indole-3-butyric acid (IBA) to the basal medium increased lateral shoot formation and ease of rooting. Microcuttings repeatedly subcultured on 1.0 M IBA produced well-developed roots, a high number of axillary shoots and could be acclimatized in the greenhouse.     

In vitro propagation of cashew from young trees

Summary Regeneration of cashew (Anacardium occidentale L.) from shoot explants of young grafts of mature tree origin is described. Establishment of shoot cultures was affected by season of collection, source, and type of explant. Explants from young grafts established better than those collected from field trees, and nodal cuttings regenerated better than shoot tips. Maximum percentage bud break and minimum contamination was noticed when shoots were collected in dry months (January to May). Pre-conditioning of stock plants by hormonal spray with 6-benzyladenine (BA) and gibberellic acid (GA₃) and brief presoaking of shoots in BA had no significant effect on culture establishment. MS (Murashige and Skoog, 1962) medium with half-strength major nutrients, 2.74 mM l-glutamine, 87.6 mM sucrose, and 2.25 gl⁻¹ phytagel was ideal for culture initiation. Inclusion of 0.1% polyvinylpyrrolidone (PVP-360) in the media reduced phenolic exudation. Solidified media was superior to liquid medium. Sucrose/glucose as energy source was found essential in the medium and had significant effect on percentage bud break and shoot development. A repeatable axillary shoot-bud induction was obtained on the above basal medium containing thidiazuron (TDZ) alone and in combination with BA. TDZ at 0.45 μM was best for axillary shoot-bud proliferation (4.5 buds per shoot) with maximum response (100%). Bud elongation could be stimulated in multiple shoots on medium containing 116.8 mM sucrose. In vitro rooting on auxin media and pulsing microshoots in 10 mM naphthalenaacetic acid (NAA) was ineffective. Rooting inability was, however, overcome by a micrografting procedure.     

Regeneration of whole plants from apical meristems of Pinus radiata

A methodology to regenerate whole plants of Pinus radiata from apical meristems of 3- and 7-year-old trees was developed. Meristematic domes with two or three leaf primordia were excised from surface-sterilized branch tips of field-grown plants and cultured in LP medium with half strength macronutrients (1/2 LP) and full strength micronutrients. The early growth of meristems required approximately 12 weeks, including a recovery stage during the first 2 weeks. After 8 weeks, some meristems developed abnormal phenotypes and died during the subsequent stages of development. However, healthy meristems elongated and formed shoots when they were transferred to LP medium supplemented with MS vitamins, 30 mg l^–1 casein hydrolysate, and 0.4 g l^–1 agar plus 2.85 g l^–1 Gelrite. Meristems that developed vigorous shoots were used for rooting experiments when they were 2 cm in length. Whole plants were obtained after 5 days of root induction in water-agar medium containing 8.2 M IBA and 5.4 M NAA and 1 month culture in LP medium with 10 g l^–1 sucrose. Plants regenerated from meristems were further propagated by rooting of cuttings. Of the rooted cuttings, 10% were morphologically juvenile.     

Regeneration of plantlets from mature embryos of western larch

Summary Larix occidentalis Nutt. can be micropropagated using whole excised mature embryos. The basal medium for induction (over 70% response) was half-strength Quoirin and LePoivre salts with Schenk and Hildebrandt organics and N⁶-benzyladenine singly or with kinetin at a final concentration of 10μM. Bud elongation was best on half-strength Schenk and Hildebrandt or Bornman’s mineral salts, and elongated shoots could be maintained on either half-strength Quoirin and LePoivre or Schenk and Hildebrandt formulations containing 0.05% activated charcoal, 2% sucrose, and 0.7% agar. Axillary buds developed naturally on elongating juvenile shoots, but a high sucrose treatment (6%) for 2 wk enhanced the number produced. Very good rooting (80 to 100%) was obtained by pulsing shoots for 2 to 3 h in a solution of indole butyric acid (1 mM, pH 4.5–5.0), or by planting shoots in peat-vermiculite moistened with basal medium containing 5μM α-naphthalene acetic acid, pH 5.0. Rooted shoots were hardened before transfer to the greenhouse, and initially were kept at low light and high humidity after transfer to ex vitro conditions. This research was supported initially by a National Research Council of Canada, Division of Energy Contract, and subsequently by a Natural Sciences and Engineering Research Council of Canada strategic grant to T. A. Thorpe.     

In vitro response of apical bud explants from mature trees of jackfruit (Artocarpus heterophyllus)

A tissue culture technique for rapid vegetative propagation of mature jackfruit trees using apical bud cultures has been developed. Shoot-tip cultures were established on MS medium with 5–10 mm explants dissected from terminal buds of new growth from trunk. After initial culture of bud explants, one- to two-node pieces were taken from the microshoots formed and used to proliferate further axillary shoots for multiplying and maintaining shoot cultures. Benzyladenine and kinetin (4.5–9.0 µM), either separately or together, supported shoot proliferation; higher concentrations of the cytokinins inhibited bud breaking and favoured callus formation at the explant bases. Bud explants taken from emerging trunk sprouts invariably produced clumps of multiple shoots, whereas buds obtained from actively growing top branches generally elongated to form a solitary shoot. November to January was the best season for initiation of cultures from field-grown trees. Shoots proliferated at the initial subcultures had mature morphology and were difficult-to-root. Shoots assumed to be juvenile-like developed at the later passages and could be rooted with 60–80% success using 1/2-MS salts and 10 µM of indolebutyric acid or naphthaleneacetic acid. Regenerated plantlets were transferred to the soil and about 50% survived.     

Micropropagation of Bauhinia variegata and Parkinsonia aculeata from nodal explants of mature trees

In vitro propagation protocols were established for two leguminous trees, Bauhinia variegata and Parkinsonia aculeata. In each case axillary shoot proliferation was achieved from nodal explants from mature (6-2-8 years) trees using Murashige & Skoog's medium supplemented with 2.22–31.1 M of 6-benzyladenine. Subsequent rooting of the regenerated shoots was achieved on medium containing 2.46–14.8 M of indole-3-butyric acid. Successful transfer of the regenerants to soil has been accomplished.     

Regeneration of plantlets through organogenesis from mature embryos of jack pine

A protocol is described for the production of plantlets from mature excised embryos of jack pine (Pinus banksiana Lamb.), a conifer widely distributed in temperate North America. Shoot buds were induced on von Arnold and Erickson's or Bornman's MCM salts with 10 M cytokinin for 2 weeks, using Phytagar^® for gelling the medium. Bud development and shoot elongation required frequent subculture on MCM medium with activated charcoal and reduced inorganic nitrogen during elongation. Shoots were rooted in peat-perlite with -naphthaleneacetic acid. The protocol produces about six plantlets per embryo.     

Regeneration of Ceratozamia euryphyllidia (Cycadales, Gymnospermae) plants from embryogenic leaf cultures derived from mature-phase trees

Embryogenic cultures were induced from pinnae removed from young leaf flushes of mature-phase trees of the endangered cycad species, Ceratozamia euryphyllidia. Induction media consisted of B5 major salts, Murashige and Skoog minor salts and organics, 400 mg l^–1 glutamine, 100 mg l^–1 asparagine, 100 mg l^–1 arginine, 60 g l^–1 sucrose, 2 g l^–1 gellan gum, 4.65–13.94 μm kinetin and 4.52–9.05 μm 2,4-dichlorophenoxyacetic acid. Cultures were maintained in darkness. Embryogenic cultures were comprised of precotyledonary somatic embryos that proliferated by somatic polyembryogenesis following subculture onto medium without plant growth regulators. Somatic embryo development and maturation occurred spontaneously from proliferating cultures on medium without plant growth regulators. Somatic embryos were monocotyledonous and mature somatic embryos germinated on semisolid medium without growth regulators. Subsequent development, which included the elongation of the first leaves, occurred only after subculture onto semisolid medium without plant growth regulators containing 0.5% (wt/vol) activated charcoal and under low light intensity. The time period from explanting to plant recovery was approximately 3 years. Received: 25 September 1997 / Revision received: 16 December 1997 / Accepted: 29 December 1997     

Regeneration and characterization of plants produced from mature tobacco pollen protoplasts via gametosomatic hybridization

Summary Mature pollen protoplasts (n) isolated from kanamycin resistant plants of Nicotiana tabacum (2n = 4x = 48) were fused with somatic mesophyll protoplasts (2n) of Nicotiana plumbaginifolia (2n = 20) to produce plants. A total of 3.6·10⁶ mature pollen protoplasts were fused with 7·10⁶ mesophyll protoplasts using a PEG/Ca²⁺ method. Mature pollen protoplasts did not divide in our culture conditions, and N. plumbaginifolia protoplasts stopped dividing when the protoplast-derived colonies were transferred to a selection medium containing paromomycine (20 mg·l^-1). A total of 133 actively growing colonies were recovered on the selection medium containing kanamycin (100 mg·l^-1). Plants from twenty resulting cell lines were confirmed as hybrids (17) or cybrids (3) based on leaf and floral morphology and fertility analysis. Isozyme pattern analysis confirmed the nuclear hybrid and cybrid nature, respectively, for 2 and 3 typical gametosomatic selected plants. Root tip squashes of 6 of the gametosomatic hybrid plants revealed chromosome numbers ranging from 44 to 68; the 3 selected cybrid plants had 48 chromosomes. Evidence for organelle transmission from the mesophyll partner in the gametosomatic plants is shown. From the analysis it can be concluded that the gametosomatic fusion involving mature pollen protoplasts (n) carrying a dominant selection marker can be convenient for synthesis of either hybrids or cybrids. Such gametosomatic fusion is therefore considered as a new approach towards the production of androgenetic plants with a choosen cytoplasm.Abbreviations AAT aspartate aminotransferase - BCP bromocresol purple - EST esterase - MES 2-(N-morpholino) ethanesulfonic acid - AP acid phosphatase - PEG polyethyleneglycol - PER peroxydase     

Chemical Composition and Bioactivities of Essential Oil from Leaves of Syzygium cumini (L.) Skeels Native to Punjab,Pakistan

Herbal medicines are widely used for the treatment of different types of diseases like skin and throat infections and other diseases in developing countries. Syzygium cumini (L.) Skeels fruit, leaves and bark were used for the remedies of different diseases anciently. The aim of the present study was to evaluate the chemical profile of Syzygium cumini leaves essential oil (EO) from Punjab, Pakistan. The essential oil was isolated using hydrodistillation technique and analyzed by gas chromatography (GC) and gas chromatography‐mass spectrometry (GC/MS). Free radical scavenging capacity and antioxidant activity were assessed by using DPPH radical scavenging ability, inhibition of linoleic acid peroxidation, bleaching of β‐carotene in linoleic acid system and reducing power assays. Antimicrobial potential was assessed by disc diffusion assay and measurement of minimum inhibitory concentration (MIC) using resazurin microtiter‐plate assay. The anti‐heme biocrystallization activity of EO was also assessed. The major components (>3%) found in Syzygium cumini leaves EO were β‐farnesene (3.42 %), caryophyllenol (3.46 %), terpinen‐4‐ol (3.61 %), β‐myrcene (3.90 %), γ‐cadinene (4.09 %), fenchol (4.22 %), cis‐β‐ocimene (4.40 %) and 5‐methyl‐1,3,6‐heptatriene (4.90 %). Excellent antioxidant, antimicrobial and weak antimalarial potential was observed. It can be concluded that Syzygium cumini leaves EO has potential application for food and pharmaceutical industries.     

Optimization of agar extraction from local seaweed species,Gracilaria salicornia in Tanzania

This study aimed to develop agar extraction protocols for Gracilaria salicornia from Tanzania and investigate its physico‐chemical characteristics. A 3³ factorial experimental design was used in the extraction of agar whereby three independent variables of NaOH concentration (10, 20 and 30% w/v), alkali pre‐treatment duration (0.5, 1 and 2 h) and extraction temperatures (115, 120 and 125°C) were used to determine the optimum conditions for production of high‐quality agar. Agar yield, gel strength, sulfate content, gelling and melting temperatures were evaluated as dependent variables. The optimal condition was observed at 30% NaOH concentration, 2 h alkali pre‐treatment duration and 120°C extraction temperature. The yield, gel strength, sulfate content, gelling and melting temperatures of the agar obtained under these conditions were 26.9 ± 0.7%, 510.3 ± 16.2 g cm^?2, 0.29 ± 0.04%, 39.3°C and 88.4°C, respectively. These properties are very close to that of imported commercial agar. It was concluded that the local agar is capable of replacing imported agar for most general purposes. This offers a new possibility of using quality local agar in place of commercial agar.     

Direct shoot regeneration from leaf segments of mature plants of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> (L.) moench

Summary This is the first communication of direct shoot regeneration from fully developed leaves of potted mature Echinacea purpurea plants. Shoot buds were induced directly on the adaxial surface of mature leaf tissues of E. purpurea 30 d after culture initiation on Woody Plant Medium (WPM) supplemented with various levels of 6-benzyladenine (BA). Maximum shoot organogenesis, with 12–20 shoots per leaf segment, was obtained with 5% coconut milk and 2.5 mg l⁻¹ (6μM) BA in 30 d. Callus was induced using 0.5 mgl⁻¹ (1μM) α-naphthaleneacetic acid and 2.5 mgl⁻¹ (6μM) BA. The regenerated shoots were rooted on WPM supplemented with 1.5 mgl⁻¹ (3μM) of indole-3-butyric acid, 3% sucrose, and 0.85% agarose. Rooted plants were successfully transferred to soil in pots and appeared morphologically normal and flowered in a growth chamber.     

Regeneration of <Emphasis Type="Italic">Prunus salicina</Emphasis> Lindl (Japanese plum) from hypocotyls of mature seeds

Plant regeneration of Prunus salicina (Japanese plum) using mature seeds was studied and evaluated. Shoots were effectively induced from hypocotyl slices of mature seeds on media containing cytokinins. Among three plant growth regulators evaluated, thidiazuron (TDZ) was the most effective for shoot induction overall. Shoots were also induced using 6-benzylaminopurine (BA), but the effectiveness was reduced at low concentrations. Low regeneration was induced using kinetin. Three plum varieties were evaluated and the regeneration appeared to be genotype dependent. Induced shoots elongated, roots formed, and plantlets developed upon transfer of the shoots to the rooting medium. Primary shoots, when sub-cultured on fresh induction medium, produced multiple shoots, and such multiplication could continue for more cycles. The plantlets were transferred to soil, and the full plants were readily recovered in a greenhouse. The regeneration process was relatively fast as plants could be recovered in 4 to 5 mo. after the culture initiation.     

Influence of alkali treatment on agar from Gracilaria cornea from Yucatán, México

The effect of alkali treatments on the yield, rheological and chemical properties of agar from Gracilaria cornea growing along the Yucatáncoast were studied in order to evaluate its potential for industrial use inan attractive economic standpoint. Alkali treatment was carried out with NaOH concentrations of 0.5%, 1%, 3% and 5% in a water bath at 80, 85 and 90 °C. Agar yield, gel strength, gelling and melting temperatures, sulphate, 3,6-anhydro-galactose and ash content weredetermined. The different combinations of NaOH concentration and treatment temperature strongly influenced agar characteristics. There was a variation in the agar content for all NaOH treatments and temperature combinations, ranging between 14.5% to 22.1%. Although the yields obtained for 0.5% NaOH at all temperatures and 1% NaOH at 80 and 85°C were higher than those required by the industry, the physical and chemical characteristics of the agar were similar to those obtained fornative agar from the same species. The gel strengths, sulphate content and gelation hysteresis obtained with agar from the 1% NaOH treatment at 90 °C are in the range required by the food industry. Treatments with 3% and 5% NaOH at all temperatures improved significantly the agar quality giving higher gel strengths (974–1758 g cm ^-2) than those reported for other Gracilaria species. This revised version was published online in August 2006 with corrections to the Cover Date.     

Rapid clonal propagation of three mulberries,Morus cathayana HemsL,M. lhou Koiz. andM. serrata Roxb., through in vitro culture of apical shoot buds and nodal explants from mature trees

High-frequency bud break and multiple shoots were induced in apical shoot buds and nodal explants ofMorus cathayana, M. lhou andM. serrata on Murashige and Skoog (MS) medium containing 0.5–1.0 mg/l 6-benzylaminopurine (BAP). Addition of gibberellic acid (0.4 mg/l) along with BAP induced faster bud break both in apical shoot buds and nodal explants and also enhanced the frequency of bud break in all three species. Shoot culture initiation was greatly influenced by explant type, explant age and explanting season. The shoots were successfully rooted on half-strength MS medium containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/l. The plantlets were successfully acclimated and eventually established in soil.Abbreviations BAP 6-Benzylaminopurine - GA ₃ Gibberellic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - IPA Indole-3-propionic acid - Kn Kinetin - MS Murashige and Skoog (1962) medium - NAA 1-Naphthalene acetic acid     

High frequency <Emphasis Type="Italic">in vitro</Emphasis> propagation of <Emphasis Type="Italic">Holarrhena antidysenterica</Emphasis> from nodal buds of mature tree

An in vitro method for propagation of Holarrhena antidysenterica Wall. has been developed using nodal explants from mature trees growing in the field. Irrespective of concentrations and combinations of growth regulators used, the axillary and terminal buds sprouted and elongated when inoculated on Murashige and Skoog (MS) medium. The highest numbers of shoots were formed when sprouted shoots were subcultured from MS basal medium onto MS medium containing 2 mg dm⁻³ N⁶-benzyladenine (BA) and 0.5 mg dm⁻³ α-naphthalene acetic acid (NAA). The shoot number further increased upon subculture on MS medium containing 0.5 mg dm⁻³ BA. By repeated sub-culturing of shoots derived from nodal axillary buds, a high frequency multiplication rate was established. The elongated shoots were excised and rooted in auxin free MS basal medium. Ex vitro rooting of in vitro formed shoots was achieved upon dipping the microshoots for 2 min in 2 mg dm⁻³ of indole-3-butyric acid solution. Successful field establishment and high (80–90 %) survival of plants was observed.