High Frequency Induction of Somatic Embryoid of Rice in Suspension Culture

On MS and B5 basic media the mature seed of rice (Oryza sativa L.) was induced to produce somatic embryoid. The results showed that the rate of occurrence of somatic embryoid varied with the variety of rice and the concentration of 2,4-D was important to the occurrence of somatic embryoid. For those varieties of rice suitable for induction, the induction factors such as KT, zeatin, BAP, ABA, osmotic pressure and so forth were stringently required at different stages of somatic embryoid development, and they also influenced the formation of somatic embryoid. The results of various layered cultures of embryonic calli proved that the somatic embryoid was derived from the surface layer callus containing embryonic cells. At the induction stage no somatic embryoid was formed in callus. The rate of somatic embryoid formation was over seventy per cent on liquid differentiation medium I.     

The effect of ovary-conditioned medium on microspore embryogenesis in common wheat (Triticum aestivum L.)

Microspores were isolated from wheat (Triticum aestivum L.) spikes of various genotypes following an effective pretreatment that induced microspore embryogenesis. The isolated microspores were cultured with or without live ovaries, and with or without medium pre-conditioned by ovaries for varying periods of time. Live ovaries alone increased androgenic embryoid yields up to 4.5-fold over the control for microspores isolated from responsive genotypes. While live ovary co-culture alone was not effective for microspores isolated from recalcitrant genotypes, the addition of medium preconditioned by ovaries to microspore cultures increased embryoid yield by more than 100-fold. Without ovary-conditioned medium, no embryoids could be obtained from some recalcitrant genotypes. Ovary-conditioned medium apparently functions to increase embryoid yields by providing essential substance(s) for elaboration of the embryogenic program already triggered during pretreatment.     

Prospects of androgenetic induction in <Emphasis Type="Italic">Lupinus</Emphasis> spp.

Anthers cultures of six Polish cultivars of pasture lupin (Lupinus L.) were examined for their androgenic response. Anthers with microspores at the uninucleate stage were isolated from flower buds and cultured in liquid media. Better viability of androgenetic structures was obtained when donor plants had grown under field as opposed to greenhouse conditions. A density of five anthers per 0.5 ml medium was more conducive to androgenetic induction than 25 anthers per 0.5 ml medium. Addition of 5% maltose to the induction medium and culture at 25°C without pre-treatment of flowers, buds or anthers promoted microspore release and division. The greatest frequency of androgenic callus, ~70% was developed from cvs. Katon, Wat (white lupin), in contrast to cvs. Legat, Juno (yellow lupin), Polonez and Sonet (narrow-leafed lupin) with callus induction ~30–40%. Despite various combinations of media tested, plant regeneration was not obtained from anther derived callus.     

Interrelationship of Abscisic Acid and Gibberellic Acid in the Promotion of Callus Formation in the Abscission Zone of Citrus Bud Cultures

The mechanism of ABA-induced callus formation was studied in sterile bud cultures of Citrus [Citrus sinensis (L.) Osbeck] on defined media. ABA was found to promote callus formation in the abscission zone between the petiole and the branch while inhibiting bud growth. The promoting effect of ABA was dependent on the physiological state of the shoot from which buds were excised, and on the size of the explant. Callus formation was highest in autumn and summer (i.e. younger) buds, and lowest in older buds excised from previous summer flush. GA was only slightly active in promoting callus formation when applied separately, but showed a highly synergistic effect when applied with ABA: maximal callus formation was attained at a combination of 10^?5M ABA and 10^?6 MGA in the medium. Subcultures of ABA-induced callus revealed that ABA inhibited the growth of isolated subcultured callus, while IAA and kinetin, and especially GA, promoted its rapid proliferation. A general decrease in protein synthesis was found in the abscission zone during the first 5 days of induction, while total protein content changed only slightly. The results suggest that ABA-induced callus formation in Citrus bud explants is a multiphasic phenomenon involving, at least, two stages: (1) activation of certain cells in the abscission zone by ABA, resulting in the formation of callus layers, and (2) subsequent proliferation of the callus tissue, which is dependent on the hormonal balance in the explant. This growth-promoting effect of ABA seems to be a general phenomenon in explants exposing a cut-surface.     

Improved somatic embryogenesis in wheat by partial simulation of the in-ovulo oxygen,growth-regulator and desiccation environments

The effects of O₂, growth-regulators and desiccation on callus growth and somatic embryo (embryoid) development were investigated in cultures of immature embryos of two lines of Triticum aestivum L. Callus and embryoid formation were induced on media that contained N⁶-furfurylamin-opurine (kinetin) and either 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-o-anisic acid, either with or without abscisic acid (ABA). Cultures containing differentiated embryoids were then exposed to high concentrations of both ABA and indole-3-acetic acid, after which samples were desiccated to approx. 10% tissue moisture. Incubating cultures in 3.2 mmol·l^-1 O₂ (approx. 9%, low-O₂) increased embryoid formation sixfold in one wheat line and nearly threefold in another. In the former line low-O₂ caused the formation of mostly embryogenic callus. Low-O₂ also decreased precocious germination of immature embryos, decreased callus growth, and improved development and viability of the resultant embryoids. Including 1.9 mol·l^-1 ABA in the callus-induction medium reduced germination of immature embryos and reduced the incidence of embryoids with visible abnormalities. Despite the improved morphology, significantly fewer of the embryoids produced on ABA-containing medium germinated. Desiccation significantly enhanced germination of these embryoids as well as those produced on ABA-free medium.Abbreviations ABA abscisic acid - DPA days post-anthesis - dicamba 3,6-dichloro-o-anisic acid - 2,4-D 2,4-dichlorophen-oxyacetic acid - FW fresh weight - IAA indole-3-acetic acid - Kin kinetin (N⁶-furfurylaminopurine) - MS Murashige and Skoog (1962) medium Contribution of the Utah Agricultural Experiment Station, Utah State University, Logan, UT, Journal Paper No. 3565     

荷兰芹胚性愈伤组织诱导及胚状体发生与内源IAA和ABA关系的初步研究

分析了荷兰芹胚性愈伤组织发生的条件, 对Co²⁺作用下胚状体形成与培养物内源IAA和ABA 的关系做了研究。结果表明, 荷兰芹下胚轴胚性发生能力随其相对位置而异, 自下而上逐渐提高。提高2, 4-D 浓度有利于胚性愈伤组织的诱导, 水解酪蛋白对胚性能力的表达没有显著影响。在胚状体发生过程中, 添加Co²⁺显著降低培养物内源IAA 和ABA 水平, 并提高胚状体诱导率。其中对照组IAA 有一个峰值, ABA 有两个峰值;实验组ABA 变化趋势与对照组相似, 而IAA 则始终没有峰值出现。Co²⁺对IAA 和ABA 的抑制机制可能有所不同。     

Accumulation of Phenolic Compounds in Conifer Callus Cultures in Response to Wood Blue-Stain Fungi

Callus cultures of Siberian larch (Larix sibiricaLedeb.) and Siberian spruce (Picea obovataLedeb.) were used to demonstrate the elicitor activity of two ophiostomatoid fungal species, Ceratocystis laricicolaand Ceratocystis polonica, as the pioneer settlers on larch and spruce, respectively. The extract from C. laricicolamycelium stimulated the accumulation of lignin in larch cells by 37% and that of bound proanthocyanidins by 25%. In spruce callus cultures, C. laricicolaand C. polonicaincreased the bound PA content by 25 and 46%, respectively. In the callus cultures of larch and spruce, the addition of extract of C. laricicolaincreased the concentration of p-hydroxybenzoic acid 13-fold and nearly 4-fold, respectively. The metabolic characteristics of accumulation of phenolic compounds in conifer cells are discussed.     

Studies on anther cultures of tomato —Lycopersicon esculentum Mill

The anthers of three genotypes ofLycopersicon esculentum, viz. cv. HS-101, cv. HS-102 and an F₁ hybrid (Montfavet 63-4xHS-101) in different stages of development were cultured in various defined nutritive media. Only anthers containing microspores in the early uninucleate stage were found to respond with the culture medium in the formation of androgenic callus. The DGII medium with 2 mg l⁻¹ NAA and 1 mg 1⁻¹ kinetin was found to be best for callus induction but MS medium supplemented with 2 mg l⁻¹ 2,4-D and 0.1 mg 1⁻¹ BAP favoured proliferation and growth of the callus. The androgenic microspores followed the ‘B’ type pathway of androgenesis in the formation of callus. Induction of tracheids in the callus could be achieved by supplementing the basal medium with NAA and kinetin or 2,4-D and BAP. Initiation of vessel elements and cambium were favoured by addition of NAA and kinetin and that of the phloem in the presence of 2,4-D and BAP in the basal medium, suggesting that the hormonal requirements for production of different elements of the vascular system in androgenic callus are different. Although roots could be induced from the callus, shoot differentiation could not be achieved under cultural conditions.     

Callus cultures and bark from Norway spruce clones show similar cellular features and relative resistance to fungal pathogens

In field experiments, clones of Norway spruce [Picea abies (L.) Karst.] showed different degrees of resistance against pathogenic fungi inoculated into the bark that correlate with differences in polyphenolic parenchyma (PP) cells of the bark. Cells of spruce callus cultures, particularly towards the callus surface, resemble PP cells and this study looks at changes in callus cells during infection and the relative resistance of cultures from clones of low (weak) or high (strong) resistance to fungal infection. Callus cultures, initiated from trees with different resistance, were co-inoculated with Ceratocystis polonica (Siem.) C. Moreau and Heterobasidion annosum (Fr.) Bref. Callus cells from strong clones resemble PP cells of bark tissue from strong clones, having more polyphenolic bodies, while callus cells from weak clones are more similar to PP cells from those clones, which have less extensive phenolic bodies. Callus cultures from trees with weak resistance were more quickly overgrown by both species of pathogenic fungi than cultures from trees with strong resistance. Callus cells of infected cultures showed changes similar to activated PP cells of bark, including enhanced accumulation of polyphenolics. Phenolic bodies were more numerous and more extensive (larger and denser) in callus cells of strong versus weak clones under all conditions. Thus, callus cells may perform similar functions in defense as PP cells in the bark. Callus from trees of varying resistance seem to reflect the relative resistance of the trees from which they are derived, and this study indicates that some mechanisms of resistance can be studied using callus from trees of different resistance.     

Endogenous ABA concentration and cytoplasmic membrane fluidity in microspores of oilseed rape (Brassica napus L.) genotypes differing in responsiveness to androgenesis induction

Key message

A better understanding of androgenesis with a focus on the changes in plasma membrane fluidity and endogenous ABA content affecting embryogenesis induction in microspore suspension of B. napus.

Abstract

Changes in plasma membrane fluidity (MF) and ABA content associated with androgenesis induction were under the study. Both parameters were monitored in microspores of two Brassica napus L. genotypes differing in their response to androgenic induction under heat (1 day at 32 °C). MF was assessed by DPH method. ABA content was evaluated by ELISA. Heat caused microspores’ plasma membrane to become more rigid. Lower MF in microspores of ‘DH 4079’ (of high androgenic potential) seems to maintain proper cell protection and leads to efficient embryogenesis induction. Plasma membrane remodelling coincided with changes of ABA content in microspores and in the culture medium in both genotypes. ABA concentration (μM) and ABA content (fmol per 10⁴ microspores or pmol g^?1 FW) were for the first time measured in microspores. ABA concentration (μM) in microspores and in the culture medium (nM) differed significantly for the genotype and the treatment. The interaction between both variables was also significant. In general, ABA content ranged from <3.5 to 87.1 fmol per 10⁴ microspores. The highest content of ABA was detected in ‘DH 4079’ microspores at 32 °C. Assuming a mean microspores’ radius of 10 μm, it corresponds to ABA concentration of 2.1 μM. Heat shock resulted in quantum of medium pH reduction (0.1–0.2) and increased levels of ABA in microspores and in the medium of both tested genotypes. However, heat induced increase of ABA content in microspores of non-responsive ‘Campino’ had no clear-cut impact, on androgenesis induction efficiency, which suggests a more complex mechanism of process initiation.     

In vitro development of plants from microspores of rice

Summary Rice (Oryza sativa L., 2n=24) anthers containing microspores in the early-uninucleate to first-mitosis stages were induced successfully to develop into plants in vitro through an intermediary step of callus formation. Callus initiation occurred with highest frequency in anthers containing mid-uninucleate microspores. The callus derived from different stages of microspore development differed in the potential to differentiate into plants. The plants regenerated from pollen callus were predominantly haploid or diploid; polyploid and aneuploid plants were relatively infrequent. The first division of the uninucleate microspores was asymmetrical, resulting in the formation of large vegetative and small generative nuclei. The vegetative nucleus divided repeatedly and assumed the major role in the formation of callus, whereas the generative nucleus degenerated rapidly. Simultaneous division of the two nuclei was observed in a few pollen grains. Nuclear fusion during the very initial stages of pollen development was postulated to account for the occurrence of the diploid and polyploid plants. This work was supported by the National Science Council, Republic of China.     

Cytochemical studies of callus development from microspore in cultured anther of rice

Cytochemical studies of androgenic anthers of Oryza sativa picked from the culture at 2 day intervals from 0 to 40 days have been carried out. Glutaradehyde-OsO₄-fixed and plastic-embedded sections were stained with TBO, SBB and PAS for acidic polymers, lipids and polysaccharides respectively. Among the population only 4% of microspores, which accumulate abundant amorphous lipid in the first few days of culture, are androgenic. Less than 30%, which have many lipid granules and some amorphous lipid, become nutritive microspores. Starch grains also accumulate in these nutritive microspores which degenerate at the stage when the androgenic multicellular microspores are in rapid development. The remaining microspores, which have no or little lipid, degenerate early. At about the 100-cell stage, each multicellular unit consists of two cell types, large and small. The large cells contain abundant amorphous lipid and starch grains which the small ones stain intensely with TBO.Our results indicate that the epidermis and endothecium of the cultured anthers are not quiescent. They can accumulate and transport lipid and polysaccharides at certain stages during the cultural period. Globular embryoid appearing structures and leaf-like protrusions can be observed at the surface of the callus in about 40-day old culture, indicating that both embryogenesis and organogenesis may take place in rice callus.Abbreviations OsO₄ osmium tetroxide - TBO toluidine blue O - SBB sudan black B - PAS periodic acid-Schiff's reagent - NAA 1-naphthaleneacetic acid     

Induced androgenesis in alfalfa (Medicago sativa L.)

Summary Anthers of 10 alfalfa (Medicago sativa L.) lines were used as initial material for the production of androgenic haploids. More than 30 variants of nutrient media were tested. Twenty five different treatments with low temperatures and gamma rays were tried in order to find optimal conditions for callus induction and organogenesis.The genotype, stage of microspore development, phytohormonal composition of the nutrient media and pretreatment with physical agents, alone or in combination, affected the efficiency of organogenesis and regeneration in anther cultures of alfalfa.Plants exhibited a high degree of variability in their chromosome number. Haploids, dihaploids and mixoploids were obtained.Cytological studies of in vitro pollen development revealed the origin of the regenerants from microspores.Abbreviations BAP 6-Benzylaminopurine - 2-ip 6-(,-dimethylallylamino)Purine - IAA Indolylacetic Acid - NAA Naphthaleneacetic Acid - 2,4-D Dichlorophenoxyacetic Acid - CMS Cytoplasmic Male Sterility     

Development of microsporesin vivo andin vitro inBrassica napus L.

Summary Populations of highly homogeneous uninucleate and binucleate microspores ofBrassica napus cv. Topas were obtained by bud selection and percoll fractionation. The development of the uninucleate and the binucleate microspores in culture was compared to thosein vivo using the fluorochrome DAPI to stain DNA. The major developmental pathway of the uninucleate microsporesin vitro resulted in embryo formation. The characteristic of this pathway was that the first division produced two diffusely stained nuclei and subsequent divisions gave rise to a multinucleate embryoid. The second pathway which occurred in a small number of the uninucleate microspores led to callus formation. The majority of the binucleate microsporesin vitro followed the developmental pattern of their counterpartsin vivo and were not embryogenic. The embryogenic binucleate microspores produced embryos through the divisions of the vegetative nucleus.Plant Research Centre Contribution # 1147     

Effect of abscisic acid on heat stress tolerance in the calli from two ecotypes of <Emphasis Type="Italic">Phragmites communis</Emphasis>

Dune reed (DR) and swamp reed (SR) are two ecotypes of reed (Phragmites communis Trin.) that displayed differences in stress tolerance. To uncover the molecular basis for such difference, the effects of heat stress were studied using the calli derived from the two ecotypes. Heat stress caused increased ion leakage, inhibited growth, decreased cell viability, and elevated hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents in the calli of both ecotypes, but DR callus showed better heat tolerance than SR callus. In DR callus, heat stress caused significant increase in the endogenous ABA content but not in SR callus. Application of fluridone (an ABA synthesis inhibitor) aggravated the heat stress damages on the DR callus whereas it had only minimal impact on the SR callus. Exogenous application of ABA alleviated the heat stress symptoms in the calli of both ecotypes. ABA treatment increased the activities of superoxide dismutase, catalase, ascorbate peroxidase and peroxidase, and also decreased H₂O₂ and MDA contents. These results indicate that the ability of ABA synthesis under heat stress is a key factor attributing to the higher heat tolerance of DR than SR.     

Callus culture from hypocotyls of Kosteletzkya virginica (L.) seedlings

It was shown that callus established from Kosteletzkya virginica (L.) Presl. (Malvaceae) can grow in salinities higher than 200 mM NaCl if previously accomodated stepwise. Callus lines developed from seedlings of different harvests or of the same harvest at different times, all showed the same pattern of growth and sensitiviy to salinity. The absorption of Na⁺ into the callus increased with increasing external NaCl concentration. In the callus, Na⁺ was apparently distributed outside and inside a cellular membrane (possibly the plasmalemma). This membrane was, apparently, capable of regulating the Na⁺ concentration in the protoplast. Outside this membrane Na⁺ accumulated to concentrations higher than in the external growth medium. Exogenously supplied proline or glycine-betaine did not affect the growth of the callus. Externally applied ABA stimulated growth under saline conditions and increased the accumulation of proline. Growth and proline content were positively correlated in callus exposed to salinity, but in the presence of ABA they were negatively correlated. ABA was involved in both growth and proline accumulation, but there was no clear relationship between these two effects. Both ABA and proline, if added to the growth medium, improved the appearence of the callus.Abbreviations ABA abscisic acid - B₅ Gamborg's medium - BA benzylalanine - 2,4-d 2,4-dichlorophenoxy acetic acid - FW fresh weight - G B₅ medium without growth regulators - GH B₅ medium supplemented with growth regulators - NAA naphthalene acetic acid - PGR plant growth regulators - Q _T total amount of a certain ion in the tissue - Q _s amount of the ion that has leaked out - QAC Quaternarty Ammonium Compounds - RGR mean relative growth rate - W₁ and W₂ fresh weight at times t₁ and t₂     

Effect of various growth regulators on growth in vitro of cherry shoot tips

An investigation was undertaken to examine the effects of nine different growth regulators on growth in vitro of shoot cultures of the semi-dwarfing cherry rootstock Colt (Prunus avium × P. pseudocerasus). The effects of each supplement on shoot extension and proliferation and also leaf and callus production were noted, and it was found that BAP has the ability to proliferate shoots, IBA nullifies this effect and that kinetin, ABA, GA₃ and ethylene inhibit the growth of colt cultures. Conditions were established which resulted in a) optimum shoot growth prior to subsequent rooting or grafting; b) maximum shoot proliferation for rapid clonal multiplication and c) minimum shoot growth. This study will form the basis of an investigation into germplasm conservation of temperate fruit trees by both cryogenic storage and minimal growth techniques.     

Production of 8-epi-tomentosin by plant cell culture ofXanthium strumarium

This study was conducted to establish a plant cell culture system for the production of medically important secondary metabolites fromXanthium strumarium. The effects of plant growth regulators including NAA, 2,4-D, kinetin, and ABA were examined in terms of callus induction, maintenance of callus and suspension cultures. It was shown that callus was induced upon treatment with NAA while embryo was induced after treatment with 2,4-D. Callus formation was further improved by treatment with ABA and NAA. The level of callusing increased by 17–29% for the seed case, cotyledon, leaf, and hypocotyl and by 96% in the case of the root. Suspension cell lines were established using calli produced from cotyledon, hypocotyl and root and cultured at 25°C under light conditions. The cells grew up to 15 g/L with NAA 2 ppm, BA 2 ppm, and ABA 1 ppm treatment. Supernatants of suspension cultures of cell lines derived from coyledon and hypocotyl produced some distinctive secondary metabolites, one of which was identified as 8-epi-tomentosin, which belongs to the xanthanolides. The amounts of 8-epi-tomentosin produced by the cotyledon-and hypocotylderived cell lines were 13.4 mg/L and 11.0 mg/L, respectively.