Periodic cooling of bird eggs reduces embryonic growth efficiency

For many bird embryos, periodic cooling occurs when the incubating adult leaves the nest to forage, but the effects of periodic cooling on embryo growth, yolk use, and metabolism are poorly known. To address this question, we conducted incubation experiments on eggs of zebra finches (Taeniopygia guttata) that were frequently cooled and then rewarmed or were allowed to develop at a constant temperature. After 12 d of incubation, embryo mass and yolk reserves were less in eggs that experienced periodic cooling than in controls incubated constantly at 37.5 degrees Celsius. Embryos that regularly cooled to 20 degrees Celsius had higher mass-specific metabolic rates than embryos incubated constantly at 37.5 degrees Celsius. Periodic cooling delayed development and increased metabolic costs, reducing the efficiency with which egg nutrients were converted into embryo tissue. Avian embryos can tolerate periodic cooling, possibly by adjusting their physiology to variable thermal conditions, but at a cost to growth efficiency as well as rate of development. This reduction in embryo growth efficiency adds a new dimension to the fitness consequences of variation in adult nest attentiveness.     

The effect of temperature on embryo survival and development in pallid sturgeon Scaphirhynchus albus (Forbes & Richardson 1905) and shovelnose sturgeon S. platorynchus (Rafinesque, 1820)

The thermal response of pallid sturgeon Scaphirhynchus albus and shovelnose sturgeon S. platorynchus embryos was determined at incubation temperatures from 8 to 26°C and 8 to 28°C, respectively. The upper and lower temperatures with 100% (LT₁₀₀) embryo mortality were 8 and 26°C for pallid sturgeon and 8 and 28°C for shovelnose sturgeon. It was concluded that 12–24°C is the approximate thermal niche for embryos of both species. Generalized additive and additive‐mixed models were used to analyze survival, developmental rate and dry weight data, and predict an optimal temperature for embryo incubation. Pallid sturgeon and shovelnose sturgeon embryo survival rates were different in intermediate and extreme temperatures. The estimated optimal temperature for embryo survival was 17–18°C for both species. A significant interaction between rate of development and temperature was found in each species. No evidence was found for a difference in timing of blastopore, neural tube closure, or formation of an S‐shaped heart between species at similar temperatures. The estimated effects of temperature on developmental rate ranged from linear to exponential shapes. The relationship for rate of development to temperature was relatively linear from 12°C to 20°C and increasingly curvilinear at temperatures exceeding 20°C, suggesting an optimal temperature near 20°C. Though significant differences in mean dry weights between species were observed, both predicted maximum weights occurred at approximately 18°C, suggesting a temperature optimum near 18°C for metabolic processes. Using thermal optimums and tolerances of embryos as a proxy to estimate spawning distributions of adults in a river with a naturally vernalized thermal regime, it is predicted that pallid sturgeon and shovelnose sturgeon spawn in the wild from 12°C to 24°C, with mass spawning likely occurring from 16°C to 20°C and with fewer individuals spawning from 12 to 15°C and 21 to 24°C. Hypolimnetic releases from Missouri River dams were examined; it was concluded that the cooler water has the potential to inhibit and delay sturgeon spawning and impede embryo incubation in areas downstream of the dams. Further investigations into this area, including potential mitigative solutions, are warranted.     

DNA ploidy abnormalities in rabbit preimplantation embryos are not increased by conditions associated with in vitro culture

Possible adverse effects of in vitro culture-associated physical factors were studied in 3- and 4-day-old rabbit embryos. Laboratory conditions were mimicked by exposure to visible light (320–740 nm, 1600 lx) or decreased temperature (22 ± 1°C). Embryos were exposed for a 24-hr period followed by either immediate evaluation or an additional 24 hr of standard in vitro culture (darkness, 37°C) and evaluation thereafter. Effects were assayed by cytophotometric measurement of the DNA content in Feulgen-stained cell nuclei and by cell number. The incidence of DNA aneuploid embryos and DNA aneuploid cell nuclei per embryo, as well as the average nuclear DNA content, was not significantly different between exposed embryos and controls. Both in vitro culture and reduced temperature caused a decrease in cell number. The temperature-induced cell number decrease was reversible within 24 hr after return to 37°C. These results demonstrate that physical factors associated with in vitro culture do not increase DNA ploidy abnormalities in cultured preimplantation embryos. Mol. Reprod. Dev. 50:30–34, 1998. © 1998 Wiley-Liss, Inc.     

The feminizing effect of metabolic heating in Green Turtle (Chelonia mydas) clutches in the eastern Mediterranean

Metabolic heating has been poorly investigated in eastern Mediterranean coastline of Turkey, which host some of the most important Green Turtle (Chelonia mydas) nesting sites in the Mediterranean. We studied the effects of clutch size and embryo numbers on nest temperature and discuss the feminizing effect of metabolic heating. Two test sites were conducted in Sugözü Beaches (Turkey). Data loggers were placed in eight nests with different clutch sizes. Nest temperature was strongly correlated with embryo numbers and metabolic heating produced by embryos was calculated to be 0.019°C per late stage embryo and 0.020°C per hatchling. Metabolic heating was calculated to be 0.6°C in the middle third of the incubation period during which sex is determined. It was estimated that metabolic heating increased 10.4% of female hatchlings. The heat produced by embryos should be taken into consideration while estimating sex ratios indirectly by nest and sand temperatures. Additionally, the metabolic heating value should be known for conservation measures, such as nest relocation, dividing the nest for controlling nest temperature, especially related to climate change.     

Influence of ration level and rearing temperature on hepatic GHR1 and 2, and hepatic and intestinal TRα and TRβ gene expression in late stages of rainbow trout embryos

The study examined whether the early life-history temperature experience of rainbow trout Oncorhynchus mykiss embryos affects subsequent growth and expression of growth-related genes in the growing-up juveniles in response to variations in ration levels. Embryos were reared in a Heath incubator at either 8·5° C (E_8·5) or 6·0° C (E_6·0) until hatching, at which time they were transferred to grow-up tanks supplied with water at 8·5° C. At swim-up, the late stage embryos were subsequently fed a salmonid starter diet at levels of 5, 2 or 0·5% of live body mass per day. The body mass and proximate composition of the juveniles was examined when yolk absorbance was complete (21 days after the fish commenced feeding). Quantitative RT-PCR was used to examine the expression of mRNA encoding for growth hormone receptors 1 and 2 (GHR1 and GHR2) in the liver, and the two isoforms of thyroid hormone receptor (TRα and TRβ) in the liver and intestinal tract. Final body mass and total length, liver and intestinal masses, and total lipid content of the E_8·5 treatment group were directly related to increased ration size. These variables in the E_6·0 treatment group fed the 5% ration were significantly lower than for the comparable E_8·5 treatment group, suggesting an effect of embryo rearing temperature on the subsequent growth of these late stage embryos as they undergo the transition from embryo to early juvenile. Intestinal TRα and TRβ mRNA abundance was directly related to ration size in the E_8·5 treatment group, but not in the E_6·0 treatment group. Conversely, hepatic TRα and TRβ mRNA abundance was significantly affected by ration size only in the E_6·0 group, with TRβ and TRα abundance showing direct and inverse relationships with ration size, respectively. Hepatic GHR1 mRNA abundance was significantly and directly related to ration size in both the E_8·5 and E_6·0 treatment groups, but there were no differences in the abundance of hepatic GHR2 mRNA among any treatments.     

Equilibrium,quasi-equilibrium,and nonequilibrium freezing of mammalian embryos

The first successful freezing of early embryos to −196°C in 1972 required that they be cooled slowly at ∼1°C/min to about −70°C. Subsequent observations and physical/chemical analyses indicate that embryos cooled at that rate dehydrate sufficiently to maintain the chemical potential of their intracellular water close to that of the water in the partly frozen extracellular solution. Consequently, such slow freezing is referred to as equilibrium freezing. In 1972 and since, a number of investigators have studied the responses of embryos to departures from equilibrium freezing. When disequilibrium is achieved by the use of higher constant cooling rates to −70°C, the result is usually intracellular ice formation and embryo death. That result is quantitatively in accord with the predictions of the physical/chemical analysis of the kinetics of water loss as a function of cooling rate. However, other procedures involving rapid nonequilibrium cooling do not result in high mortality. One common element in these other nonequilibrium procedures is that, before the temperature has dropped to a level that permits intracellular ice formation, the embryo water content is reduced to the point at which the subsequent rapid nonequilibrium cooling results in either the formation of small innocuous intracellular ice crystals or the conversion of the intracellular solution into a glass. In both cases, high survival requires that subsequent warming be rapid, to prevent recrystallization or devitrification. The physical/ chemical analysis developed for initially nondehydrated cells appears generally applicable to these other nonequilibrium procedures as well.     

Peroxidases in Relation to Removal of Dormancy and Germination of Apple Embryos

The changes in germination, peroxidase activity and isoperoxidase spectrum have been studied in apple embryos at 5°C (stratification) and at 20°C in the presence or absence of seed coats. The embryo dormancy is progressively released at 5°C, but not at 20°C. The peroxidase activity in embryos covered with seed coats is very low at 5°C as well as at 20°C which corresponds to a restricted number of isoenzymes. In isolated embryos the peroxidase activity increases significantly. This is due to an increase in both the number and the activity of the isoperoxidases and it is more pronounced at 20°C than at 5°C. The obtained results suggest that the soluble peroxidases are not involved in the process of the release of embryo dormancy. The variations observed are attributed to the growth process following germination, which can occur even at low temperature.     

Freezing mammalian embryos: A review of the techniques

This article reviews the literature on freezing mammalian oocytes and embryos, with emphasis on embryos of domestic animals. Mammalian embryos must be stored in a quiescent state to retain viability for long periods. This has been accomplished by freezing and storing the embryos at ?196°C. To freeze embryos, a cryoprotectant like dimethyl sulfoxide (DMSO) or glycerol was required, slow cooling (0.1 to 2.0°C/min) and warming (1 to 50°C/min) rates were used, enucleation or seeding the freezing medium was a necessity, and stepwise addition and removal of the cryoprotectant at room temperature seemed to be beneficial. Using the above parameters embryos have been frozen and stored at ?196°C for several years and upon thawing and transfer to a suitable recipient, viable offspring have developed. Initially embryo viability was low after freezing-thawing, but with refinement of freezing-thawing techniques has increased sufficiently so that freezing embryos is no longer a laboratory technique, but is applicable to field use.     

Plant regeneration from microspore-derived embryos ofBrassica Napus: Effect of embryo age,culture temperature,osmotic pressure,and abscisic acid

Summary Microscope cultures ofBrassica napus cv. Topas undergo high frequency embryogenesis in vitro; however, the majority of microspore-derived embryos do not develop directly into plants but usually undergo abnormal development including the formation of secondary embryos on the hypocotyls. The present studies show that older embryos or embryos cultured at higher temperature (25° C) were more likely to undergo secondary embryogenesis whereas embryos cultured at 20° C or pretreated at 5° to 10° C for 28 days developed more readily into normal plants. Compared with embryos cultured at 25° C, those cultured at 20° C gave a threefold increase in normal plant production. Pretreatments at cooler temperatures (5° to 10° C) resulted in an additional two-to threefold increase in the recovery of normal plants. Higher osmoticum during pretreatment improved embryo survival at low temperatures but generally inhibited normal plant development. Abscisic acid was ineffective or deleterious.     

Incubation temperature critical to successful stimulation of in vitro zygotic embryo growth in four Australian native Cyperaceae species

Many species of Western Australian Cyperaceae (sedges) are vital components of the indigenous flora but commonly display low seed set, poor seed quality and intractable seed dormancy. We report the effects of incubation temperature and in vitro growth media on whole seed germination compared with extracted zygotic embryo growth in Tetraria capillaris, T. octandra, Lepidosperma drummondii and L. tenue. No germination was observed from intact whole seeds of all test species regardless of the treatment evaluated. In contrast, excised zygotic embryos of all study species exhibited significant increases in growth when cultured at 15°C compared to embryos incubated at 25°C; however, optimal media for embryo growth were genera specific. Extracted embryos of T. capillaris and T. octandra exhibited maximum percentage growth (30 and 40%, respectively) at 15°C on ½ MS medium with no plant growth regulators required. In the case of L. drummondii and L. tenue 1 μM thidiazuron was a necessary addition to the ½ MS medium resulting in 40 and 77% growth of embryos (at 15°C), respectively. Incubation of extracted embryos at 25°C (regardless of medium treatment) resulted in <10% embryo growth for T. octandra and L. tenue, while the remaining two species (L. drummondii, T. capillaris) showed no embryo growth at 25°C on any medium treatment.     

Effect of plant growth conditions, plating density, and genotype on the anther culture response of soft white spring wheat hybrids

Ten soft white spring wheat (Triticum aestivum L.) F₁ hybrids were grown under three temperature regimes, and anthers were cultured at two plating densities to investigate the effect of plant growth conditions, plating density, and genotype on embryo induction and plant regeneration. Anthers from plants grown at high temperature (25 °/18 °C) or from plants transferred from low (15 °/12 °C) to high temperature generally produced more embryos and green shoots, with a lower frequency of albinos, than did anthers from plants grown at low temperature. However, plating densities of 10 versus 20 anthers per milliliter, had little effect on anther response. Four of the five hybrids with `Fielder' as the female parent produced more embryos and green shoots than did hybrids with `AC Reed' as the female parent. Received: 12 July 1996 / Revision received: 1 April 1997 / Accepted: 30 April 1997     

Effects of low temperatures on in vitro produced bovine zygotes

The objective of this research was to investigate the effects of cooling on the development of bovine zygotes. One-cell bovine embryos were maintained at 39°C (control), 20°C, 10°C, or 0°C for 5, 10, or 20 minutes, then cultured in vitro for 7 days and the proportion of embryos developing to the compact morula or blastocyst stage compared between different treatments. Duration of exposure time had no effect on development. Development rates to the compact morula or blastocyst stage were 3.9%, 11.4%, 17.4%, and 24.4% for zygotes maintained at 0°C, 10°C, 20°C, and 39°C, respectively, with differences in embryo yield between every treatment (P < 0.05). In a second experiment, bovine pronuclei (karyoplasts) and cytoplasts were cooled at 0°C or maintained at 39°C for 5 minutes. Pronuclear transplantation was then utilized to create 4 types of reconstructed embryos, those with: 1) non-cooled pronuclei and non-cooled cytoplasm, 2) non-cooled pronuclei and cooled cytoplasm, 3) cooled pronuclei and non-cooled cytoplasm, and 4) cooled pronuclei and cooled cytoplasm. The proportion of embryos developing to the blastocyst stage was highest when non-cooled pronuclei were transferred into non-cooled cytoplasm (18.9%), and similar to that of non-cooled, non-manipulated control zygotes (13.2%, P > 0.05). No embryos developed to the blastocyst stage when pronuclei (cooled or non-cooled) were transferred into cooled cytoplasm. However, zygotes with cooled pronuclei transferred into non-cooled cytoplasm yielded 4.5% blastocysts (P < 0.05). More embryos developed to the compact morula or blastocyst stage when non-cooled vs. cooled cytoplasm was utilized, regardless of whether the pronuclei were cooled (P < 0.05). These data demonstrate that pronuclei are more tolerant to low temperature exposure than is ovum cytoplasm. Mol. Reprod. Dev. 47:435–439, 1997. © 1997 Wiley-Liss, Inc.     

Growth of swimming muscles and its metabolic cost in larvae of whitefish at different temperatures

The temperature relationship of routine metabolic rate (R_r) of non-feeding, non-growing Coregonus lavaretus larvae between 2 and 15°C is characterized by Q₁₀-values ranging from l.8-2.45. The rate of growth, based on weight determinations, of first-feeding larvae amounted to 3.5, 7.6 and 9.4% day-¹ at 5, 10 and 12°C respectively, from which Q₁₀-values between 4.0 and 4.8 can be calculated. The rate of increase of muscle mass between 5 and 10°C, based on the determination of the cross-sectional area of inner muscle fibres, resulted in a Q₁₀-value of 4.5. Water temperature influenced the pattern of growth of the inner muscle fibres. At hatching, after 360 day degrees, total muscle mass of larvae reared at 4 and 8°C was independent of temperature, but at 4°C the rate of mass increase owed more to hyperplasia (increase in fibre number) than to hypertrophy (increase in fibre mass), whereas at 8°C the opposite was the case. The calculation of power budgets (including the metabolic cost of growth) of first-feeding larvae yielded net conversion efficiencies (K₂) increasing with temperature from 46.3% at 5°C to 54.7% at 12°C. Comparing our data with literature data two general conclusions can be drawn. (1) In first-feeding larvae the net, but not the gross, conversion efficiency of food energy increases with temperature. This is due to net energy input being characterized by a much higher Q₁₀-value than energy expenditures. (2) In embryos of freshwater fish so far investigated hyperplasia plays a greater role in the increase of fibre mass than hypertrophy at the lower temperature, whereas in embryos of marine fish hyperplasia prevails at the higher temperature. It is suggested that this discrepancy correlates with the high concentration of free amino acids in the eggs of marine species which provide an additional, easily available, source of metabolic energy absent in freshwater species.     

Growth and reproduction of the mosquitofish,Gambusia affinis,in relation to temperature and ration level: consequences for life history

The allocation of energy to growth and reproduction, in relation to temperature and food availability, was investigated in laboratory experiments with the mosquitofish,Gambusia affinis. At constant temperature of 20, 25 and 30°C and ad libitum feeding, specific growth rates increased with increasing temperature at 1.7, 3.1 and 3.4% dry mass day⁻¹, respectively. Growth rates in a cycling temperature regime (20–30°C, ) were faster than in a 25°C constant temperature. As temperature increased from 20 to 30°C, mean age at first reproduction decreased from 191 to 56 days and brood size and mass of offspring increased significantly. Interbrood interval was also temperature dependent; estimates at 25 and 30°C for females >1000 mg were 22.6 and 18.6 days, respectively. Interbrood interval could not be calculated at 20°C. Although fitness was highest at 30°C, females at 25°C invested a greater proportion of surplus energy (growth and reproduction) to reproduction (38%) than at 20 (17%) or 30°C (36%) during the 32-week study. Fish at cooler temperatures began reproduction at a smaller size. Where rations were controlled at low, medium, and ad libitum levels, somatic and gonadal growth increased with increasing temperatures and food availability. The proportion of energy invested in reproduction was highest at 25°C for each comparable ration level. Calculated energy budgets indicated that over the 10-week study, 17–22% of the food energy was invested in growth, 0–7% in reproduction, and 75–83% in respiration and excretory losses, depending on feeding and temperature conditions.     

Seed dormancy and germination in Jeffersonia dubia (Berberidaceae) as affected by temperature and gibberellic acid

The genus Jeffersonia, which contains only two species, has a trans‐Atlantic disjunct distribution. The aims of this study were to determine the requirements for breaking dormancy and germination of J. dubia seeds and to compare its dormancy characteristics with those of the congener in eastern North America. Ripe seeds of J. dubia contain an underdeveloped embryo and were permeable to water. In nature, seeds were dispersed in May, while embryos began to grow in September, and were fully elongated by late November. Germination started in March of the next year, and seeds emerged as seedlings soon after germination. In laboratory experiments, incubation at high temperatures (25 °C, 25/15 °C) for at least 8 weeks was required to initiate embryo growth, while a transfer to moderate temperatures (20/10 °C, 15/6 °C) was needed for the completion of embryo growth. At least 8 weeks at 5 °C was effective in overcoming physiological dormancy and for germination in seeds after the embryos had fully elongated. Thus, both high and low temperatures were essential to break dormancy. Gibberellic acid (GA₃) treatment could substitute for the high temperature requirement, but not for the low temperature requirement. Based on the dormancy‐breaking requirements, it is confirmed that the seeds have deep simple morphophysiological dormancy. This dormancy type is similar to that of seeds of the eastern North American species J. diphylla. Although seeds require 10–11 months from seed dispersal to germination in nature, under controlled conditions they required only 3 months after treatment with 1000 mg·l^?1 GA₃, followed by incubation at 15/6 °C. This represents practical knowledge for propagation of these plants from seed.     

Emergence of sexual size dimorphism and stage‐specific effects of elevated temperature on growth rate and development rate in Harmonia axyridis

Temperature is considered to be the most important environmental factor influencing the performance of ectotherms because it determines the rate of most biochemical reactions and thus the efficiency of metabolism and its function. Unfortunately, most studies investigate the effects of temperature on individuals exposed to a particular temperature regime during their whole pre‐imaginal development and detailed information on variation of the temperature effects during ontogeny is rare. In the present study, the effects of the timing of exposure to a transient period of elevated temperature during ontogeny on development rate and growth rate are investigated for the ladybird Harmonia axyridis Pallas. Control beetles are reared at a constant temperature of 20 °C, whereas treated beetles are reared at 20 °C but are exposed to 33 °C for 48 h either during the early‐larval stage, third‐larval instar, fourth‐larval instar or the pupal stage. The rate of development and the growth rate are both accelerated because the timing of exposure to elevated temperature occurred later in pre‐imaginal development (i.e. development rate and growth rate are highest in individuals exposed to elevated temperature during the pupal stage). An exception to this pattern is the lowering of development rate in individuals exposed to elevated temperature during the fourth‐larval instar. Female H. axyridis have a significantly higher development rate and growth rate than males. However, the relative difference in growth rate between the sexes is much higher than the difference in development rate between sexes, resulting in a female‐biased size (mass) dimorphism in adult beetles.     

A novel method of cryopreservation without a cryoprotectant for immature somatic embryos of conifer

Cryopreservation of embryogenic tissue is an essential storage step in genotype selection and seedling production through somatic embryogenesis. To date, immature conifer somatic embryos, at the proliferation step, were only able to tolerate ultra low temperature after prior cryoprotectant treatments. We report a novel cryopreservation method for conifer (interior spruce and Douglas-fir) embryogenic tissue focusing on the maturation step of developing embryos that forgoes such cryoprotectant treatment. In this study, somatic embryos matured on culture media containing abscisic acid (ABA) at 20°C for 8 weeks. Typically, matured embryos in this manner were able to survive cryopreservation. The embryogenicity, however, decreased with increasing embryo maturity. Non-freezing low temperatures, such as 5°C, not only inhibited cotyledon development but also maintained embryogenicity. Cryotolerance was successfully induced when embryos were matured (or pretreated) under 5°C for a suitable culture period, typically 4–8 weeks. These embryos were able to survive a rapid cooling process and liquid nitrogen storage without the addition of any cryoprotectants. After cryopreservation, embryogenic tissue was recovered in both interior spruce and Douglas-fir. Embryo maturation tests indicated no difference in mature embryo yields with or without cryopreservation in interior spruce. The key factors inducing cryotolerance included ABA supplementation in culture media and low temperature pretreatment. Optimum combinations of these factors can result in high rates of tissue survival and high embryogenicity after cryopreservation.     

Factors affecting the dormancy and germination of bleeding heart [Lamprocapnos spectabilis (L.) Fukuhara] seeds

• Information on the optimal conditions to promote the germination of Lamprocapnos spectabilis (L.) Fukuhara seeds is limited; consequently, this study was conducted to establish the requirements to break seed dormancy and promote germination.
• The selected seeds had morphophysiological dormancy and had not begun embryo development. To study the dormancy breaking and embryo development processes, seeds were subjected to constant or changing temperature treatments during moist stratification.
• High temperature and humidity resulted in vigorous embryo growth, with the longest embryos occurring after 1 month of incubation at 20 °C. At 4 °C, the seeds required incubation period of at least 3 months to germinate. Embryo growth and germination were higher with changing high and low temperatures than under a constant temperature, and changing temperatures also considerably changed the endogenous hormone levels, embryo development and germination. Bioactive gibberellin (GA) content was higher in seeds incubated at 20 °C for 1 month, then at 4 °C for 2 months. The content of endogenous abscisic acid in seeds subjected to the same treatment decreased by 97.6% compared with that of the untreated seeds.
• Embryo growth and seed germination require changing high and low temperatures; however, exogenous GA₃ could substitute for high temperatures, as it also causes accelerated germination. In this study, the seeds of L. spectabilis were identified as an intermediate simple type, a sub‐level of morphophysiologically dormant seeds.

     

Influence of freezing and non-freezing temperature on somatic embryogenesis and vinblastine production in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don.

Catharanthus roseus (L.) G. Don. (Apocynaceae) is an important dicotyledonous medicinal plant. It produces vinblastine and vincristine, two alkaloids that are being used against a variety of cancers. In the present study, the freezing (−196, 4, 15°C) and non-freezing (25°C) temperature was imposed on embryogenic cultures, and later in vitro embryogeny and vinblastine production in C. roseus was studied. Somatic embryo (SE) production was maximum at 15°C, but the SE maturation was high at 4°C. The SEs, grown at 25°C, showed highest germination and plantlet conversion. Quantitative estimation of vinblastine was carried out using high-performance liquid chromatography in various in vitro raised tissues (embryogenic callus), embryo stages (proliferated, matured and germinated embryos)], and SE-derived plantlets (leaf, shoot, root and whole plant) after various freezing- and non-freezing temperature treatments. Vinblastine synthesis was temperature dependent in C. roseus that has been discussed in this present article.