Rate Of Embryogenesis Research Articles

TOWARDS THE CLONAL PROPAGATION OF COCONUT

Past studies from our laboratory have shown that whole immature, or mature sliced, zygotic embryos are a very good starting explant for coconut somatic embryogenesis. The highest rate of somatic embryogenesis was obtained when certain polyamines were added into the culture medium as well as activated charcoal (AC) to absorb unwanted phenolics. These past studies also showed that the development and maturation of the somatic embryos produced could be improved by the addition of abscisic acid (ABA), alone or with one of several osmotically active agents, into the culture medium. In the present study this well characterised somatic embryogenic system for zygotic tissues is being modified and applied to somatic tissues. This recent approach should be a better method for the rapid production of clonal, true-to-type coconut palms. The present research approach is focused on young leaf section explants which have been found to be very responsive to callus production. Young leaf sections produced optimum callus when cultured on media containing 2,4-D (150 μM) and the amount produced could be increased by soaking the sections in sterile water (15 to 60 minutes) or ascorbic acid (15 to 30 minutes) prior to culturing. Further improvement in callus production, as well as a reduction in the time taken for callogenesis was obtained when casein hydrolysate and/or certain polyamines were added to the callus induction medium. The development of the somatic embryos was improved by using ABA and polyethylene glycol (PEG) in the maturation medium. Despite these initial successes in improving coconut somatic embryogenesis, further studies are now being considered to shorten the time to achieve somatic embryogenesis, to better germinate somatic embryos and to improve the rate of somatic seedling conversion into plantlets.

Toxicity of Hg, Cu, Cd, and Cr on early developmental stages of Ciona intestinalis (Chordata, Ascidiacea) with potential application in marine water quality assessment

The toxicity of mercury, copper, cadmium and chromium on sperm viability, fertilisation, embryogenesis and larval attachment of Ciona intestinalis was examined. Fertilisation rate (FR) showed a small decrease even at the highest metal concentration tested. The median effective concentrations (EC 50) reducing rates of embryogenesis and larval attachment by 50% were 54 μg Hg/l (0.27 μM), 46 μg Cu/l (0.72 μM), 838 μg Cd/l (7.46 μM), 10,318 μg Cr/l (198 μM), and 35 μg Hg/l (0.18 μM), 34 μg Cu/l (0.54 μM) and 11,755 μg Cr/l (226 μM), respectively. Therefore, Hg is three times more toxic than Cu (on a molar basis), ca. 30 times more toxic than Cd and ca. 1000 times more toxic than Cr to early stages of C. intestinalis. Rates of larval attachment and embryogenesis were the most sensitive endpoints, although the latter is more advisable for routine assessment of seawater quality because of its greater simplicity. In addition to bivalves and sea-urchins, ascidian embryos can provide biological criteria for seawater quality standards taking into account the sensitivity of a chordate and contributing to the detection of harmful chemicals with no marked effect on the species currently in use in seawater quality bioassays.

グラジオラス木子へのガンマ線照射がカルス形成，不定はい形成および再生個体の花色変異に及ぼす影響

木子茎頂からの不定胚培養系を用いて, 'トラベラー', 'トパーズ'における木子へのガンマ線照射がカルス形成, 不定胚形成および再生個体の花色変異に及ぼす影響を検討した.不定胚を形成したカルスの割合は, 両品種とも照射線量が増加するに従って低下した.カルスおよび不定胚形成に対する木子へのガンマ線照射線量のRD50は, 線量率10Gy・hr-1で100∿200Gyであった.'トラベラー'には花色変異体が生じたが, 'トパーズ'ではまったく生じなかった.'トラベラー'においては培養単独よりもガンマ線照射を併用することで花色変異体の出現頻度が増加した.また培養単独よりも濃色化の程度の高い個体および形態変異した個体が出現した.さらに花色変異体には, 従来問題となっていた区分キメラがほとんどなかった.これらの結果から, 木子にガンマ線を照射し, 培養する方法は, 培養を単独で行うよりも変異を質的, 量的に拡大すること, および花色変異が完全突然変異体で生じることから, 育種上有効であると考えられる.

THERMAL ECOLOGY AND SEXUAL SIZE DIMORPHISM IN NORTHERN WATER SNAKES,NERODIA SIPEDON

We used more than 326 000 observations of temperature collected by radio telemetry from 38 individuals over three years to investigate thermoregulation and thermal relations of northern water snakes (Nerodia sipedon) near the northern limit of their distribution in Ontario, Canada. We tested hypotheses concerning the effects of feeding, season, sex, and reproductive condition on thermoregulation of individuals. The mean preferred body temperature (PBT) for captive snakes from the study population was 27.1°C, similar to that reported for other populations, and PBT range (defined as the 25th–75th percentiles of selected temperatures) was 25–30°C. When environmental conditions allowed, the mean and range of body temperature (Tb) of free-living snakes were nearly identical to those observed in captivity. The typical daily pattern in snake Tb was an increase in late morning to a plateau temperature in the preferred range, followed by a decrease in the evening with a nightime plateau at approximately the temperature of the water. We calculated indices of thermoregulation to relate Tb data to available ambient temperatures. Despite the northern latitude of the study population, ambient conditions were favorable over much of the activity season and offered the potential for the snakes to maintain Tb within the PBT range almost 24 h per day for much of the season. However, the thermoregulation indices indicated that N. sipedon are only moderate thermoregulators, and often do not exploit opportunities to achieve PBT, particularly during the day. Variation in annual ambient temperatures affected growth rates of the snakes, and was sufficient to affect the age of maturity of females. Feeding did not elicit a thermophilic response under laboratory conditions or when snakes were fed experimentally in the field, suggesting that the benefits of increasing Tb after eating did not outweigh the costs. Reproductive females thermoregulated more carefully than nonreproductive females during the July–August gestation period. Pregnant females may increase thermoregulatory behavior to enhance the rate and efficiency of embryogenesis. Males thermoregulated less than both reproductive and nonreproductive females, particularly later in the activity season when thermoregulation by females increased. We hypothesized that males pursue a survival strategy following the spring mating season and thus remain cool and secretive during the later part of the activity season. This strategy by males clearly contributes to their slowed growth and smaller size relative to females.

The Effect of Light and Auxins on the Regeneration of Lily (Lilium RegaleWil.) Cells by Somatic Embryogenesis and Organogenesis

Somatic embryogenesis and organogenesis of lilies have not been investigated in detail before, although tissue culture methods have been applied to the propagation for decades. We investigated the effects of light and different auxins on regeneration in lily (Lilium regale Wil.) callus at a morphologic and anatomic level. In addition to solid medium, cell suspension cultures were established for somatic embryogenesis. Our results showed a high regeneration capacity for lily callus and a rate of somatic embryogenesis that reached 10% of all shoots formed. Differentiation was promoted when the callus was kept in the dark during propagation and transferred into light only prior to regeneration. Naphtalene acetic acid was superior compared with 2,4‐D in both the regeneration and proliferation of callus of this species, as 2,4‐D resulted in malformed shoots and necrosis, and it inhibited the regeneration in general. Morphological investigations shared differences in the structure of bulbets arising from the different regeneration processes. Callus in suspension showed high regeneration capacity, and organogenic and embryogenic sites and somatic proembryos were detected.

ジャーファーメンター型培養槽によるウドの懸濁培養細胞からの不定はい誘導

ウドの品種'愛知坊主'の懸濁培養細胞から効率的に不定胚誘導を行うため, 培養細胞の継代培養条件と数種類のジャーファーメンター型培養槽により誘導される不定胚の数と形状について検討した.不定胚形成能の高い培養細胞は, 0.5 mg・liter-1の2, 4-Dを添加したMS液体培地(30 g・liter-1ショ糖添加)を用い, 3週間ごとに継代することで維持できた.各種培養槽で誘導された不定胚数は, 培地量100ml当たりエアーリフト型と回転ドラム型でいずれも約189個, 気泡塔型では約124個得られた.また, 水平回転培養により三角フラスコ(100ml容)で得られた不定胚数は, 各培養槽と比較して単位容量当たり3.5∿5.3倍多く得られた.しかし, 誘導された不定胚の形状をみると, 正常胚の割合は各培養槽とも95%以上と高かったが, フラスコでは66.3%と低かった.これらのことから, エアーリフト型または回転ドラム型の培養槽を用いると形状の良い不定胚が効率的に誘導できることが明らかとなった.

In vitro development of early proembryos and plant regeneration via microculture in Oryza sativa

Two convenient and efficient microculture techniques (liquid droplet and shallow-layered culture) were used to rear 2-day-old and 3 to 4 day-old proembryos in rice. Among four cultivars, growth rate and frequency of embryogenesis were higher in the japonica cultivars than in the indica cultivars during proembryo culture. Two-day-old proembryos could grow and form callus only in Km8p and N6 among four kinds of tested media, and plantlets regenerated via organogenesis. Plant regeneration from callus initiated from three- and four-day-old proembryos occurred through somatic embryogenesis and organogenesis. For in vitro embryogenesis it was essential to supplement the medium with 14 amino acids and coconut milk. The highest frequency of embryogenesis and the frequency of total induction after 14 days of culture were approximately 42% and 95% for 3-day-old proembryos, and 45% and 100% for 4-day-old proembryos, respectively.

Juvenile hormone synthesis in relation to corpus allatum development in embryos of the viviparous cockroach Diploptera punctata

Summary Few studies have addressed endocrinology of the corpora allata (CA) in insect embryos. We now report on development and biosynthetic activity of CA in embryos of a viviparous cockroach, Diploptera punctata. When newly-eclosed adult females of D. punctata were mated, they oviposited and gave birth, respectively, about 8 and 73 days later; thus, gestation and corresponding embryogenesis lasted approximately 65 days. Dorsal closure, which coincides with differentiation of the CA, was concluded when embryos were about 13 days old and had completed 20% of embryogenesis. Reverse phase-high performance liquid chromatography revealed that embryonic CA released predominantly juvenile hormone III (JH) in vitro. Furthermore, an in vitro radiochemical assay showed that between day 28 of embryogenesis (43% of embryonic development completed) and hatch rates of JH synthesis rose, plateaued and then fell. When CA activity was increasing or was high, from day 28 to 54 (83% development), mitosis occurred at low and constant rates within embryonic CA, and corpus allatum cell number increased gradually. Between days 56 (86% development) and 60 (92% development), CA activity fell to a low level, rates of mitosis peaked, and corpus allatum cell number rose rapidly. Throughout embryogenesis, CA volume increased in parallel with CA cell number, suggesting that glandular growth was due largely to cell proliferation. Although CA activity and volume changed considerably in embryos, the diameter of corpus allatum cells, as measured from enzymatically dissociated CA, remained surprisingly constant at 11–12 μm on days 32, 46 and 60 (49, 71 and 92% development). Ultrastructural observations confirmed the large size of cells in low-activity CA of 60-day-old embryos and also showed that these cells, like those in highly active CA of 46-day-old embryos, contained abundant cytoplasm, ribosomes, microtubules and mitochondria.

Regeneration of plantlets from NaCl tolerant callus lines of Cymbopogon martinii (Roxb.) Wats.

Plantlets were regenerated successfully through somatic embryogenesis of NaCl stressed (up to 350 mM) and subsequently selected, tolerant (up to 300 mM) callus lines of the aromatic grass Cymbopogon martinii (Roxb.) Wats. The NaCl tolerant callus lines were isolated, characterized and selected on the basis of their growth behaviour as well as physiological parameters (K +/Na + ratio and proline contents) and compared with those of the wild type (control) callus lines. The regeneration potential of the NaCl tolerant callus ranged from 81.6 to 0% against 80.4 to 0% in the control lines with the NaCl level varying between 0–300 mM and 0–200 mM respectively in the medium. The embryogenic potential of NaCl tolerant callus selected even at 300 mM could be improved significantly by the incorporation of gibberellic acid (GA 3) and abscisic acid (ABA) in the medium where, with 2 mg/l of GA 3 and 1 mg/l of ABA the highest rates of embryogenesis (44.5, 28.8 and 18.6%) were achieved against 17.5, 8.2 and 1.8% on medium devoid of GA 3 and ABA at 50, 150 and 250 mM of NaCl respectively. Complete tissue browning and subsequent death were observed at 300 mM NaCl in the differentiation medium. Plantlets regenerated from NaCl tolerant callus were transferred to soil and grown under green house conditions with about 70% transplantation success.

Somatic embryogenesis in mature zygotic embryo culture of Glehnia littoralis

In vitro somatic embryogenesis of Glehnia littoralis Fr. schm. was observed when zygotic embryos were cultured on a medium containing 1-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid (0.01-10 μM), with 1 μM being the optimum. Microscopic observations revealed globular, heart-shaped and torpedo-shaped embryo formations and plantlet regeneration. These somatic embryos seemed to be produced directly from cells of the zygotic embryos used as explants. Of seven types of media tested, Nitsch's medium showed the highest rate of somatic embryogenesis. Somatic embryos developed into normal plantlets and were able to be potted.

Embryogenesis induction in petals of Araujia sericifera

The embryogenic capacity of Araujia sericifera petals and some of the factors involved in the induction of embryos was investigated. The influence of 6-benzyladenine and α-naphthalene acetic acid, light intensity (90 or 5 µmol m-2 s-1) and silver thiosulphate (inhibitor of ethylene action) were studied. It was found that petals are an easy system in which to induce somatic embryogenesis. Plants were recovered from somatic embryos. Although 6-benzyladenine is essential for inducing an efficient response, a high dosage increased callogenesis and reduced embryogenesis. The highest rate of embryogenesis is induced with high light intensity (90–100 µmol m-2 s-1), even though the presence of silver thiosulphate in the medium markedly reduced embryo induction.

Effect of12C+5 ion beam irradiation on cell viability and plant regeneration in callus, protoplasts and cell suspensions ofLavateva thuringiaca

The biological effects of irradiation with(12)C(+5) ion beam on plant cells have been analyzed. Protoplasts and cell suspensions ofLavatera thuringiaca, and a somatic hybrid callus (Hibiscus rosa-sinensis +Lavatera thuringiaca), were irradiated with doses from 0.05 to 50 Gy, and the effects on cell growth, cell division, cell viability and embryogenesis rates were analyzed. Irradiation with(12)C(+5) ion beam at relatively very low doses (5.0 Gy) significantly inhibited cell division, yet the survival rate and regeneration capability of the cells through somatic embryogenesis were conserved in more than 70 and 50 %, respectively. These results indicate that cell division is the most sensitive parameter to irradiation, accounting for the inhibition of colony formation and callus growth. The potential use of the(12)C(+5) ion beam in asymmetric protoplast fusion experiments is discussed.

Maternal Manipulation of Offspring Phenotypes via Nest‐Site Selection in an Oviparous Lizard

Mothers may be able to manipulate the phenotypes of their progeny not only via direct pathways (e.g., allocation of nutrients and energy), but also indirectly, by inducing particular developmental pathways through selection of nest sites and thus, incubation conditions. In the field, female skinks (Bassiana duperreyi) in montane southeastern Australia select nest sites with specific thermal characteristics–especially, a high mean temperature and a high diel range in temperature. Monitoring of 14 natural nests throughout most of the incubation period revealed considerable differences among nests in both means and variances of thermal regimes. Laboratory experiments on this species show that both the mean and the variance of incubation temperatures profoundly influence developmental rates (and hence, incubation periods) as well as the body shape, activity levels, thermo—regulatory behavior, and running speeds of the hatchlings. Hence, a female's selection of a particular nest site can substantially modify both the time of emergence of the hatchlings, and the morphology and behavior of her offspring. Even within a single nest, minor differences among eggs in their depth below the ground surface will directly affect thermal variance, and so may strongly influence rates of embryogenesis and the phenotype of the hatchling. The effects of thermal variance (independent of the mean) on embryonic development rates and hatchling phenotypes suggest that data from constant—temperature incubation in the laboratory should be interpreted with caution

The effects of 2ip and 2,4-D on rice calli differentiation

The role of isopentenyl adenine (2ip) in cereal tissue and plant regeneration was analyzed by addition of this compound into various cultural media. Preculture of rice calli in the presence of certain levels of 2ip before transferring onto a normal tissue culture regeneration medium increased significantly the rate of embryogenesis as well as plant regeneration. Different combinations of 2ip and 2,4-D in preculture media had profound effects on cell differentiation. The procedures of embryogenesis and plant regeneration can be separated by manipulating the ratio of 2,4-D to 2ip in the culture media. Various possible functions of 2ip, 2,4-D and KT in cereal regeneration are also suggested.

The identification of the optimum conditions for the embryonation ofToxocara canisova

Suspensions of unembryonated Toxocara canis ova were maintained at 24°C, 30°C and 37°C in solutions of 0.4% and 1.6% and 3% formalin. At temperatures between 24°C and 30°C an increase in the rate of embryonation was observed especially from the morula to the early motile embryo stages. At 37°C, embryogenesis failed to develop. The rate of embryogenesis was found to be slower the higher the fixative concentration became. Over time, a decline in the percentage viabilities of the embryonated ova was also found as the fixative concentration increased. It seems that the fastest means to produce embryonated ova is to maintain them in a solution of 0.4% formalin at 30°C and exposed to light. However, after this process, in order to ensure the long term survival of these ova, it appears that it is best to store them at 24°C or less and ensure that a sufficient concentration of dissolved oxygen is available.

High efficiency adventive embryogenesis on somatic embryos of anther, filament and immature proembryo origin in horse-chestnut (Aesculus hippocastanum L.) tissue culture

Adventive embryogenesis was successfully induced in cultures of zygotic and somatic embryos on MS medium supplemented with BA and NAA. A procedure has been proved successful for the in vitro multiplication of somatic embryos regenerated at low frequencies from filament and callus cultures. The occurrence and rate of adventive embryogenesis did not depend on the origin of the primary embryos (zygotic and somatic), but did depend on the developmental stage. Primary embryos are capable of embryogenesis in each of the different phases of embryogenesis, though the rate is different. BA concentrations of 22–44 μM increased the rate of adventive embryogenesis and accelerated the development of embryos. The highest proliferation rate (22–25x/5 weeks) was achieved at hormone concentrations of 44 μM BA and 5.4 μM NAA.

Cryopreservation of immature embryos of Theobroma cacao

Immature, white zygotic embryos of Theobroma cacao L. (cacao) retained the ability to produce callus and to undergo somatic embryogenesis after slow hydrated freezing and desiccated fast freezing in liquid nitrogen. The highest rate of somatic embryogenesis occurred in embryos which were precultured on a medium containing 3% sucrose, frozen slowly with cryoprotectants before exposure to liquid nitrogen, and recovered on a medium containing 3 mg/liter NAA. Embryos precultured on media containing sucrose increasing to 21% had a higher rate of survival but were less embryogenic after freezing. These results suggest that immature embryos might be used for long-term germplasm storage of T. cacao germplasm.

Somatic embryogenesis and plantlet regeneration ofTheobroma cacao

Abstract Somatic embryogenesis was observed on cacao (Theobroma cacao L.) expiants obtained from white to white‐pink zygotic embryos of variety ‘Amelonado’ cultured on media supplemented with napthalene acetic acid (NAA), 1–32 μM, after six weeks of culturing. A high incidence of development‐arrested somatic embryogenesis (50%) occurred with expiants cultured in Murashige and Skoog medium containing 32 uM NAA, 0.10% casein hydrolysate and 3.0% sucrose. Embryogenesis rates were different among zygotic embryo expiants from different ‘Mexico’ strains or different pods of the ‘Amelonado’ variety. The regeneration of a plantlet via direct somatic embryogenesis from the hypocotyl surface of immature cacao zygotic embryos was observed.

Somatic embryogenesis and plant regeneration from embryonic tissues ofCamellia japonica L.

Somatic embryogenesis and plantlet regeneration were achieved from immature and mature zygoticCamellia japonica embryos cultured on Murashige & Skoog's mineral medium without growth regulators or with various combinations of IBA and BAR The dependence of embryogenesis rates on growth regulator levels was not clear, though high concentrations such as 4 mg 1-1BAP plus 2 mg 1-1IBA were definitely inhibitory. BAP at 1 or 2 mg 1-1 did appear to determine the formation of ‘bud-like embryos’. By far the most responsive initial explants were immature embryonic axes collected in September, 94% of which produced somatic embryos as against only 20% for embryonic axes from mature seeds collected in October. Cotyledon explants were also embryogenic. Somatic embryos differentiating directly on the hypocotyl of the embryonic axes or the surface of cotyledons passed through typical stages of embryogenesis. Indirect somatic embryogenesis via callus was also evident. Embryogenic potential was maintained by secondary embryogenesis through the successive generations of embryos.

Embryogenesis and the effects of temperature on embryonic development, juvenile growth rates, and the onset of oviposition in the fresh-water pulmonate gastropodPhysella cubensis

Summary Embryonic development in Physella cubensis is similar to that in other members of Physella, although trochophore or veliger-like embryonic stages previously noted in P. gyrina were lacking. Egg masses of adults collected from Shades Creek, Birmingham, Alabama were subjected to three temperature treatments (10, 20 and 30°C) within the normal range of temperatures experienced in the field, and developmental stages were documented until hatching. Hatched juveniles were raised in their respective temperature treatments to sexual maturation (onset of egg mass deposition). Temperature dramatically affected the time course of embryonic development. Juveniles (0.8 mm shell length) hatched in 36 days at 10°C, 13 days at 20°C, and 9 days at 30°C. Juveniles raised at 20 and 30°C produced egg masses 69 and 35 days after hatching and had shell lengths of 6.2 and 4.4 mm, respectively. No egg masses were produced at 10°C. Body size at first reproduction was significantly smaller in the individuals raised at 30 than at 20°C indicating that somatic growth takes precedence over reproduction at lower temperatures. Juveniles raised at 10°C did not attain sexual maturity over 100 days and reached only 1.5 mm shell length. The pronounced effect of temperature on rates of embryogenesis, juvenile growth, and the onset of sexual maturation suggests temperature plays a significant role in regulating the population dynamics of P. cubensis.