Callus Induction in Small Flowered Willow Herb (<Emphasis Type="Italic">Epilobium parviflorum</Emphasis> Schreb)

In order to determine the most suitable in vitro tissue culture and plant regeneration conditions for the small flowered willow herb (Epilobium parviflorum Schreb), various explants were cultured on semi-solid MS media containing factorial combinations of plant growth regulators. Callus induction from hypocotyl, cotyledon, petiole and leaf explants was achieved on media containing 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (KIN). All other growth regulator combinations [□-naphtaleneacetic acid (NAA) ± benzylaminopurine (BAP), NAA ± thidiazuron (TDZ), indol acetic acid (IAA) ± Zeatin (ZEA)] tested failed to respond. The best results with cotyledon- and petiole- derived callus were obtained from MS medium supplemented with 1.0 mg l^?1 2,4-D + 0.1 mg l^?1 KIN and 2.0 mg l^?1 2,4-D + 0.2 mg l^?1 KIN. It was observed that B5 basal medium was more effective than MS basal medium for producing seedling and the most effective seed sterilizing solution was 25 % (v/v) sodium hypochlorite (NaOCl). No plant regeneration was observed in either callus induction or during the subculturing stage. This is the first report on in vitro tissue culture study within the genus Epilobium.     

<Emphasis Type="Italic">In vitro</Emphasis> growth profile and comparative leaf anatomy of the C<Subscript>3</Subscript>–C<Subscript>4</Subscript> intermediate plant <Emphasis Type="Italic">Mollugo nudicaulis</Emphasis> Lam.

Mollugo nudicaulis Lam., commonly known as John’s folly or naked-stem carpetweed, is an ephemeral species of tropical regions. The plant is ideal to study the eco-physiological adaptations of C₃–C₄ intermediate plants. In the present report, in vitro growth profiling of the plant and comparative leaf anatomy under in vitro and ex vitro conditions were studied. In vitro propagation of the plant was carried out on Murashige and Skoog (MS) basal medium augmented with additives and solidified with 0.8% (w/v) agar-agar or 0.16% (w/v) Phytagel?. The concentration of plant growth regulators (PGRs) in the basal medium was optimized for callus induction, callus proliferation, shoot regeneration, and in vitro rooting. The optimum callus induction was obtained from M. nudicaulis seedling hypocotyls. The highest regeneration induction of about 88% or nearly 41 shoots with about 142 leaves per culture vessel was observed from friable callus on MS basal medium solidified with Phytagel? and containing 4.44 μM 6-benzylaminopurine, 4.65 μM kinetin, 2.69 μM naphthaleneacetic acid, and 0.91 μM thidiazuron. In leaf anatomy, differences related to photosynthetic tissue organization were observed in leaves of in vitro and ex vitro plants, which indicated that changes in the environment affected the anatomy of subsequent leaves in plants. This is the first report of an efficient micropropagation protocol for M. nudicaulis, using an indirect organogenesis method. Efforts were made to optimize the concentrations of various PGRs and organic compounds for in vitro growth of regenerated shoots.     

Phytobeneficial and salt stress mitigating efficacy of IAA producing salt tolerant strains in Gossypium hirsutum

Salinity is one of the major agricultural concern that significantly limits the crop productivity. The plant growth promoting rhizobacteria (PGPR) may contribute in sustainable crop production under salt stress. The current study was designed to isolate the Indole Acetic Acid (IAA) producing salt tolerant PGPR to promote the growth of cotton (Gossypium hirsutum, FH-142) and induce its salt stress tolerance. Ten Salt Tolerant (ST) bacterial strains were screened for their PGP trait in vitro and evaluated for their beneficial effect on cotton plants growth by plant–microbe interaction assay in lab and under natural condition. GC–MS analysis of the metabolites of the selected bacterial strains confirmed the presence of indolic compounds like indole, indole-3-butyramide, benzylmalonic acid and 4-methyl-2-pyrrolidinone. The bacterial isolates ST4, ST5, ST6, ST15, ST16, ST17, ST18, ST20, ST22 and ST25 were identified as Bacillus sp., B. sonorensis, B. cereus, B. subtilis, Brevibacillus sp. B. safensis, B. paramycoides, Bacillus sp., B. cereus and B. tequilensis respectively on the basis of 16S rDNA sequencing. Bacteria inoculated plants had a significant (P < 0.05) increase in percentage germination up to (31%), root length (17%) and shoot length (34%) in lab while in wire house pot experiments, maximum enhancement in root length (31%) and shoot length (29%) was observed. ST bacterial strains inoculation improved the chlorophyll content index (34%), relative water content (36%), leaf area (33%), absorption of K⁺ (28%) and decreased the uptake of Na⁺ (58%) from soil in plants under salt stress over control in pot experiment. These ST PGPR have the potential to act as plant defense agents by enhancing plant growth, productivity, and tolerance in saline environment.     

Regeneration of plants through somatic embryogenesis in Thymus hyemalis Lange,a potential medicinal and aromatic plant

A protocol for somatic embryogenesis was developed for Thymus hyemalis, a wild species in the Mediterranean region. First, the effects of explant type, plant growth regulators [kinetin (KIN) and 2,4-dichlorophenoxyacetic acid (2,4-D)], and genotype on callus induction were tested. For callus induction, the node was the best explant; Murashige and Skoog (MS) medium supplemented with 1.8 μM 2,4-D and 0.5 μM KIN was the best medium, and the genotype had a highly significant effect. To induce production of somatic embryos, the effects of KIN, 6-benzylaminopurine (BAP), and naphthalene acetic acid (NAA) were evaluated. After 5 wk of culture in the dark, MS medium supplemented with 4.44 μM BAP, 0.54 μM NAA, and 4.65 μM KIN gave the highest percentage (85%) of embryogenic callus and the highest number of somatic embryos (27.00) per 45 mg of callus. For germination and plant recovery, somatic embryos were transferred to MS medium without plant growth regulators and plantlet conversion from developed somatic embryos was 90%. In vitro plants with adequate growth and sufficient root systems were subsequently transplanted into a mixture of peat and vermiculite (2:1?v/v) under greenhouse conditions. The survival rate of the plantlets under ex vitro conditions was 80%.     

Tissue culture of Hypericum brasiliense Choisy: Shoot multiplication and callus induction

Hypericum brasiliense, a non-domesticated plant has been shown to have useful medicinal properties. This plant has not been cultivated so a protocol for mass propagation based on selection of superior clones was developed and a protocol established for the culture of callus cells that could be used for in vitro metabolite production. A micropropagation method based on amplification of nodal buds was developed, by selection, from ten seedling clones that were examined for growth rate, multiplication rate and rooting. The effect of various basal media, growth regulator types and concentrations were examined for optimal callus induction. Optimal callus induction occured on either Murashige and Skoog's or Gamborg's media supplemented with 1 to 2 mg l^–1 of 2,4-dichlorophenoxyacetic acid.Abbreviations B5 Gamborg's medium - 2,4-Dscd 2,4-dichlorophenoxyacetic acid - IAA indolacetic acid - MS Murashige & Skoog's medium - NAA naphtaleneacetic acid     

Micropropagation of Eucalyptus benthamii to form a clonal micro-garden

Eucalyptus benthamii is an important component of forestry plantations in cold regions, but it is difficult to obtain clonal plants of this species, especially by low rooting. In this study, we developed a method for cloning selected genotypes of E. benthamii using a micropropagation technique, enabling the formation of a clonal micro-garden. Nodal segments from sprouts of mini-stumps in the clonal mini-garden were used as explants. After in vitro establishment of the explants, we tested two selected clones (BP101 and BP118), three culture media (Wood Plant Medium (WPM), Correia and colleagues JADS medium, and Murashige and Skoog medium), and two plant growth regulators (6-benzylaminopurine (BAP) and ??-naphthaleneacetic acid (NAA)) for the multiplication of adventitious buds. Additionally, combinations of two other plant growth regulators (BAP and gibberellic acid (GA₃)) were tested for the elongation of shoots. The in vitro and ex vitro rooting of micro-plantlets prior to acclimatization were compared. The in vitro bud multiplication of E. benthamii depended on the clone, culture medium, and concentration of plant growth regulators. The best results were obtained with WPM supplemented with 0.5?mg?L^?1 BAP and 0.05?mg?L^?1 NAA. The elongation of shoots depended on the clone and plant growth regulator, and the best results were obtained with nutrient medium free of GA₃ and BAP. Histological analysis showed that both in vitro and ex vitro rooting were successful, resulting in normal development of adventitious roots showing a vascular connection with the vascular cambium. The new protocol is efficient for micro-plantlet production of E. benthamii and can be used for the formation of a clonal micro-garden for other Eucalyptus or tree species.     

Micropropagation of Mandevilla moricandiana (A.DC.) Woodson

A protocol was developed for micropropagation of Mandevilla moricandiana (A.DC.) Woodson, a native plant from Brazil. Shoots, obtained from in vitro plantlets were used as source of nodal segments for shoot production from axillary buds. The nodal segments were grown on Murashige and Skoog medium supplemented with different concentrations of 6-benzyladenine and/or indole-3-acetic acid to induce axillary bud elongation. After a 2-mo culture period, the medium supplemented with 1.0 mg?L^?1 6-benzyladenine gave the largest number of nodal segments per explant. The nodal segments obtained from plants developed under these conditions were grown on medium supplemented with different concentrations indole-3-acetic acid, ??-naphthaleneacetic acid, and indole-3-butyric acid. The use of the medium supplemented with indole-3-acetic acid and indole-3-buryric induced shoot elongation and shoot development, formation of basal callus, and/or indirect organogenesis of roots. Following transfer of shoots to soil, the plants with only basal callus showed 10% survival and developed roots from callus, while in vitro-rooted plants had a maximum 40% survival rate ex vitro. Regardless of the auxin added to the rooting medium, the acclimatization period allowed the plants rooted in vitro to develop their shoots fully. The protocol developed here is suitable for the production of shoots and rooted plantlets of M. moricandiana.     

Micropropagation, <Emphasis Type="Italic">in vitro</Emphasis> flowering,and tuberization in <Emphasis Type="Italic">Brachystelma glabrum</Emphasis> Hook.f., an endemic species

Brachystelma glabrum Hook.f. is an endemic plant species of Eastern Ghats, India. In this study, efficient protocols for in vitro micropropagation, flowering, and tuberization of this plant were developed. Sterilized shoot tip and nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators (PGRs) and additives for shoot induction and multiplication. Both shoot tip and nodal explants showed the best response (90 and 100%, respectively) on MS medium supplemented with thidiazuron (TDZ) at 1.0 mg L^?1. The microshoots multiplied best on MS + TDZ (1.0 mg L^?1) in combination with α-naphthaleneacetic acid (NAA) at 0.5 mg L^?1 and coconut water (CW) at 25%. The highest number of in vitro flowers (4.0 flowers per microshoot) was observed on MS medium supplemented with a combination of N6-benzyladenine (BA) and indole-3-butyric acid (IBA), each at 1.5 mg L^?1. In vitro-derived shoots produced aerial tubers on MS + TDZ (2.0 mg L^?1) + IBA (0.5 mg L^?1) and basal tubers on MS + TDZ at 2.0 mg L^?1. In vitro shoots were best rooted on half-strength (½) MS + NAA at 0.5 mg L^?1. The rooted plantlets were successfully acclimatized in pots with 70% survival after a hardening period of 1 mo. This protocol provides an effective method for the conservation of this endemic plant species.     

Micropropagation and assessment of genetic stability in Celastrus paniculatus: An endangered medicinal plant

A highly efficient protocol for in vitro regeneration of an indigenous, endangered medicinal plant Celastrus paniculatus was achieved using nodal explants. Murashige and Skoog (MS) basal medium supplemented with 0.5 mg/L 6-benzylaminopurine (BAP) and 0.1 mg/L naphthaleneacetic acid (NAA) showed maximum percentage of shoot multiplication (83.4%) with 8.2 shoots/explants. Maximum rooting of 73.3% with 4.8 roots/shoot was achieved on half-strength MS media supplemented with 0.5 mg/L indole-3-acetic acid (IAA) and the percentage of survival was 91% after acclimatization. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) marker study confirmed genetic stability for in vitro raised explants by showing 100% monomorphism. High multiplication rate associated with genetic stability ensure the efficacy of the present in vitro clonal propagation protocol of this important medicinal plant species.     

Efficient propagation and rooting of three citrus rootstocks using different plant growth regulators

The influence of various basal medium and plant growth regulators on the efficient micropropagation of nodal explants from mature trees of alemow, sour orange, and ??Cleopatra?? mandarin citrus rootstocks was studied. All three citrus rootstock shoot cultures showed a preference for high-salt media, like Murashige and Skoog or Driver and Kuniyuki Walnut medium. Several combinations of N ⁶-benzyladenine (BA) and adenine (AD), kinetin (KIN) or gibberellic acid (GA) were tested to optimize the shoot proliferation phase. BA/GA combinations improved the proliferation of all the rootstocks studied, especially alemow. The addition of BA and AD to the culture medium improved shoot proliferation in sour orange and ??Cleopatra?? mandarin in the same way as BA and GA. The addition of different combinations of BA/KIN did not result in further improvement of any of the studied variables. The transfer of in vitro shoots to rooting media, containing different concentrations of indolebutyric acid (IBA) and indoleacetic acid (IAA), resulted in regeneration of complete plantlets. Alemow and ??Cleopatra?? mandarin shoots rooted well using these plant growth regulators; however, all combinations of IBA and IAA tested resulted in very low rooting percentages in sour orange. To improve rooting in sour orange and ??Cleopatra?? mandarin, different combinations of naphthaleneacetic acid (NAA) and IBA were tested. All NAA/IBA combinations produced higher rooting percentages than did the IBA/IAA combinations, and in sour orange nearly 100 % of explants developed roots. An efficient and simple protocol for the micropropagation of three citrus rootstocks, alemow, ??Cleopatra?? mandarin, and sour orange, by culturing nodes from mature plants, has been established.     

Factors influencing induction,propagation and regeneration of mature zygotic embryo-derived callus from Allium cepa

A systematic study on the effects of subspecies, cultivar, basal medium, sucrose concentration and 2,4-dichlorophenoxyacetic acid concentration on callus induction, propagation and subsequent plant regeneration in Allium cepa has been carried out. Mature zygotic embryos from two onion (cvs. Sturon and Hyton) and two shallot (cvs. Tropix and Atlas) varieties were used as explants. After callus initiation and growth on both Murashige and Skoog (MS) and Gamborg's B5 modified by Dunstan and Short (BDS) basal media with different 2,4-dichlorophenoxyacetic acid and sucrose concentrations for eight weeks, lines were identified on which compact or friable callus was induced. Callus induction and propagation were largely determined by the concentration of 2,4-dichlorophenoxyacetic acid whereas subspecies, cultivar, sucrose concentration and basal media were of less importance. After callus propagation for twelve weeks, 315 lines from a total of 3348 embryos initially subcultured were selected to test their regeneration capacity on growth regulator-free medium. It was found that shallot formed more shoots and roots than onion. The MS basal medium proved to be more beneficial for shoot regeneration and root formation than the BDS basal medium. There were no differences in plant regeneration among selected calli which had been previously subcultured on different concentrations of 2,4-dichlorophenoxyacetic acid and sucrose. The results show that plant regeneration strongly depended on the line: 45.4% from 315 tested lines could produce shoots while 93.0% formed roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Roles of in vitro plantlet age and growing period in the phenolic constituent yields of acclimatized Hypericum polyanthemum

The influences of culture period on growth, plant survival rate and content of phenolic compounds were investigated in vitro and in acclimatized field-grown plants of Hypericum polyanthemum. The growth kinetics of micropropagated plantlets cultured in MS modified medium and the concomitant transference to ex vitro conditions showed that cultures achieved maximum biomass and yield of bioactive compounds after 12 weeks of in vitro growth, with field-grown plants displaying the same survival pattern. Differences in yield among plants cultured in vitro for 8 and 12 weeks that were acclimatized and followed over two years showed that the physiological age of the in vitro cultures influenced biomass production. However, benzopyrans and total phenolic compounds (TPC) contents did not vary significantly, with the exception of the 5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran (HP3) concentration in the reproductive parts, which was higher in the plants grown in vitro for 12 weeks over the two years of the study. All analyzed plant parts from the spring harvest accumulated lower benzopyran levels than plants harvested after 18 weeks of growth in both treatments, except for the levels of 6-isobutyryl-5,7-dimethoxy-2,2-dimethyl-benzopyran (HP1) and 7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran (HP2) in the new vegetative parts of the plants, which did not vary. The concentration of TPC, which was detected at low levels in the old vegetative parts in both treatments, was not altered in other plant parts. The information provided by this work will help structure plant growth and collection periods designed to optimize the yield of each required bioactive metabolite.     

In vitro propagation of Western Australian Rushes (Restionaceae and related families) by embryo culture. Part 2. Micropropagation

Micropropagation of 21 species of Restionaceae and the closely relatedmonotypic families Anarthriaceae and Ecdeiocoleaceae is discussed. Multiplication rates ranged from 2-fold to 14-fold each 4–6 week subculture passage, with most species averaging 3–5-fold. The majority of taxa preferred half-strength Murashige and Skoog basal media with 1 M benzyladenine, with certain species requiring other specific treatments (e.g. Woody Plant medium). Approximately half of the species produced roots successfully (i.e. >50%) in vitro on half-strength MS with no growth regulators (or no auxins), or initiated roots after transfer to soil; other species required a longer (6–7 week) incubation on quarter-strength MS medium for initiation to occur. This paper describes the first successful micropropagation of these taxa with application for horticultural development of this important southern-hemisphere family.Abbreviations MS Murashige & Skoog (1962) basal medium - 1/4 MS quarter-strength MS (minerals only) - 1/2 MS half-strength MS (minerals only) - BA benzyladenine - WPM woody plant medium (Lloyd and McCown 1980) - GA₃ gibberellic acid - TDZ thidiazuron - IBA indolebutyric acid     

Thidiazuron enhances shoot organogenesis from leaf explants of Saussurea involucrata Kar. et Kir

An efficient protocol for the in vitro micrpropagation of Saussurea involucrata Kar. et Kir, an endangered Chinese medicinal plant, was developed. Shoot organogenesis was obtained following culture of leaf explants on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ). After 28 d of culture, 15.6?±?1.4 shoots were regenerated per leaf explant on MS medium containing 0.5 ??M TDZ. After transfer of shoots to a medium containing 5.0 ??M indole-3-acetic acid, approximately 80% of the regenerated shoots formed roots and whole plantlets. After transfer of rooted shoots to the greenhouse, 83% of the regenerated plantlets survived and grew vigorously. The regeneration protocol developed in this study provides a basis for germplasm conservation and for the production of plant material necessary to study the medicinally active components of S. involucrata.     

Direct and indirect shoot and bulblet regeneration from cultured leaf explants of Lilium pumilum,an endangered species

Alternative methods of in vitro cloning that involve both adventitious (direct) and callus intermediate (indirect) pathways were investigated for the endangered species Lilium pumilum. Plantlet regeneration was obtained from leaf explants, cultured on Murashige and Skoog (MS) basal medium supplemented with various combinations of auxins and cytokinins at different concentrations. About 30% of the explants directly formed adventitious shoots on MS medium containing 8.88 μM 6-benzyladenine (BA) and 2.69 μM α-naphthaleneacetic acid (NAA). For production of regenerable callus, callus formation followed by shoot induction was best when explants were initially cultured on MS medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Regenerable calli were yellow or purple and readily regenerated shoots when subcultured onto MS medium containing 2.22 μM BA and 1.61 μM NAA. About 78% of the calli were able to produce adventitious shoots. Shoots were rooted on half-strength MS medium supplemented with 1.34 μM NAA and were successfully acclimatized to greenhouse conditions. This report describes an efficient method for the in vitro multiplication of whole plants from leaf explants of the endangered species L. pumilum.     

Plant growth promoting effect of <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> H-2-5 on crop plants and influence on physiological changes in soybean under soil salinity

This study was aimed to identify plant growth-promoting bacterial isolates from soil samples and to investigate their ability to improve plant growth and salt tolerance by analysing phytohormones production and phosphate solubilisation. Among the four tested bacterial isolates (I-2-1, H-1-4, H-2-3, and H-2-5), H-2-5 was able to enhance the growth of Chinese cabbage, radish, tomato, and mustard plants. The isolated bacterium H-2-5 was identified as Bacillus amyloliquefaciens H-2-5 based on 16S rDNA sequence and phylogenetic analysis. The secretion of gibberellins (GA₄, GA₈, GA₉, GA₁₉, and GA₂₀) from B. amyloliquefaciens H-2-5 and their phosphate solubilisation ability may contribute to enhance plant growth. In addition, the H-2-5-mediated mitigation of short term salt stress was tested on soybean plants that were affected by sodium chloride. Abscisic acid (ABA) produced by the H-2-5 bacterium suppressed the NaCl-induced stress effects in soybean by enhancing plant growth and GA₄ content, and by lowering the concentration of ABA, salicylic acid, jasmonic acid, and proline. These results suggest that GAs, ABA production, and the phosphate solubilisation capacity of B. amyloliquefaciens H-2-5 are important stimulators that promote plant growth through their interaction and also to improve plant growth by physiological changes in soybean at saline soil.     

Effects of flask sealing and growth regulators on in vitro propagation of neem (Azadirachta indica A. Juss.)

In vitro propagation of neem (Azadirachta indica A. Juss.) may offer an efficient alternative to seed propagation of this species. For optimization of in vitro propagation, different basal salt formulations, growth regulators, and culture container sealants (polytetrafluoroethylene hydrophobic membranes [PTFE]) were evaluated. Nodal segments cultured on Murashige and Skoog (MS) medium showed the highest shoot formation per explant (1.67). Explants cultured in flasks containing MS medium with 0.5 mg L⁻¹ benzyladenine, 0.5 mg L⁻¹ kinetin, and 0.05 mg L⁻¹ naphthaleneacetic acid, and sealed with two PTFE membranes, produced the highest number of shoots (4.04). In contrast, explants cultured in flasks without membranes showed leaf chlorosis and senescence. For plant recovery, regenerants were acclimatized in a substrate of coconut fiber and eucalyptus bark (1:1) and showed 80% survival. Our results indicated that the use of flasks with vents was beneficial for in vitro propagation of this important plant.     

An efficient method for <Emphasis Type="Italic">in vitro</Emphasis> regeneration of leafy spurge (<Emphasis Type="Italic">Euphorbia esula</Emphasis> L.)

Leafy spurge (Euphorbia esula L.) is a perennial, invasive weed used as a model to study invasive plant behavior, because molecular tools (such as a deep expressed sequence tag database and deoxyribonucleic acid microarrays) have been developed for this species. However, the lack of effective in vitro regeneration and genetic transformation systems has hampered molecular approaches to study leafy spurge. In this study, we describe an efficient in vitro regeneration system. Three highly regenerative lines were selected by screening the in vitro regeneration capabilities of stem explants of 162 seedlings. The effects of various culture conditions on in vitro regeneration were then evaluated based on explant competence to form calluses and shoots. High rates of shoot regeneration can be obtained using a growth medium containing 1× woody plant basal medium and 1× Murashige and Skoog (MS) basal salts, 1× MS vitamins, 1.11 μM 6-benzylaminopurine, 1.97 μM indole-3-butyric acid, and 3% sucrose, pH 5.6–5.8. After 30 d culture, multiple shoots formed either directly from the stem or indirectly from the callus. This method is a requisite for the development of genetic transformation systems for leafy spurge and may be used to develop in vitro regeneration techniques for other species in the Euphorbiaceae.     

Development of a reproducible method to determine minimum inhibitory concentration (MIC) of plant extract against a slow-growing mycoplasmas organism

Mycoplasma species are fastidious bacteria that require a specialized medium for their growth, isolation and identification. There are no standardized tests to evaluate the in vitro susceptibility of mycoplasmas to medicinal plant extracts. A widely used in-broth, microtitre plate, minimum inhibitory concentration (MIC) assay was adapted and evaluated using acetone extracts of Anoigeissus leiocarpus on the isolates of Mycoplasma mycoides subsp. mycoides small colony variants (MmmSC). Several problems were encountered including the contamination of the medium by Bacillus species found in plants and the fact that the slow-growing mycoplasmas proved to be poor reducers of the indicator tetrazolium salt or resorcinol. We then examined a pH indicator-dependant technique to detect the acid production caused by the growth of the organism after glucose utilization from the broth medium. The method gives a clear cut-off point that was easy to read and interpret and was also reproducible.The MIC value for acetone extract of A. leiocarpus was 0.16 mg/ml. The development of this method now makes it possible to evaluate extracts of several plant species for antimycoplasmal activity.     

Somatic embryogenesis and plant regeneration in diploid Allium fistulosum × A. cepa F1 hybrid onions

Procedures were developed for disinfestation of non-dormant basal plate tissue excised from field grown basal plate tissue of diploid Allium fistulosum × A. cepa F₁ hybrid onions. Contamination levels varied with the season and vegetative development of plant material. Callus initiated from basal plate tissue and immature inflorescences of the F₁ hybrids was maintained on a BDS-based medium containing 0.75 mg/l picloram and 2.0 mg/l BA. When this medium was supplemented with vitamins and glycine, and with proline at 2.5 gm/1, somatic embryos began to form. Their development continued on a BDS-based shoot promotion medium containing 0.03 mg/l picloram and 0.32 mg/l 2iP supplemented with vitamins, glycine and proline. Genotypes differed significantly in the numbers of structures regenerated. Plantlets from somatic embryos were rooted into BDS or half-strength BDS medium without growth substances and were successfully transferred to sterilized potting mix in plastic commercial corsage boxes.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid - 2iP N6-(2-isopentenyl)adenine - NAA 1-naphthylacetic acid - BDS Gamborg's B5 medium modified by Dunstan and Short (1977a)