In vitro differentiation of embryogenic pollen,control by cold treatment and embryo formation inab initio pollen cultures ofNicotiana tabacum var. badischer burley

Summary InNicotiana cold treatment causes differentiation of embryogenic pollen. This differentiation initiates on the plant and is completed in culture. Differentiation on the plant results in pollen dimorphism and differentiation in culture leads to pollen embryogenesis. An increased number of pollen capable of embryogenesis is possible on plants induced to flower in short days and low temperature (8 hours light, 18 °C). These embryogenic pollen on selective isolation, from buds at a petal length of 3.4±0.1 cm, fail to form embryos but do so in the cultures which receive cold treatment at 10 °C for 10 days. To some extent the differentiation of embryogenic pollen can be completed on plants induced to flower at 15 °C and embryogenic pollen from such plants form embryos at a low frequency which can be substantially increased on giving the cultures a cold treatment. The frequency of embryogenesis is higher in cultures of 15 °C plants than those of 18 °C plants. Low temperature requirements at two stages—to the plant and to the culture—are essential and complimentary for embryogenesis inab initio pollen cultures.Cold treatment causes repression of gametophytic differentiation and this results in the differentiation of embryogenic potential. The embryogenic pollen, unlike gametophytic pollen, are not fully differentiated structures. They are unable to divide and form embryos in presence of metabolic inhibitors such as actinomycin-D and cycloheximide.     

Ultrastructure of embryogenic pollen ofNicotiana tabacum var. Badischer burley

Summary Pollen grains capable of embryogenesis were selectively isolated from (a) near-mature buds from plants induced to flower in short days and low temperature (8 hours light and 18 °C) and (b) young buds from these plants with an additional low temperature treatment (10 °C for 10 days) and fixed for electron microscopy. The pollen from the former formed embryos at a very low frequency in culture, and at the subcellular level showed different degrees of regression of cytoplasm and mitochondria. On the contrary, cold-treated pollen were characterized by a high frequency of embryogenesis, up to 25% of the cultured pollen. They did not show regression of cytoplasm or organelles but had an attenuated cytoplasm which was not rich in ribosomes. Another noteworthy feature of embryogenic grains was the condensed nature of mitochondria. These characteristics of embryogenic grains indicate that they are repressed for gametophytic differentiation. The embryogenic pollen did not differentiate from gametophytic pollen which were very distinctive, having a thick exine, and dense cytoplasm rich in ribosomes. The close similarity of embryogenic grains with young microspores in terms of thin exine and sparse cytoplasm is suggestive of an indeterminate state and that determination into gametophytic or sporophytic (embryogenic) type is probably the function of differential gene activity. Of interest, in this context, is the condensation of mitochondria in embryogenic grains. The relationship, if any, between mitochondrial condensation and embryogenesis remains to be resolved.     

Selection of embryogenic pollen from cold-treated buds ofNicotiana tabacum var. badischer burley and their development into embryos in cultures

Summary By using density gradient centrifugation, employing 55% percoll and 4% sucrose as suspension medium, it is possible to select embryogenic pollen from buds after cold treatment at 10 °C for 8 or more days. These buds at the uninucleate stage of pollen were collected from plants grown in 8 hours photocycles at 18 °C and supplied with mineral salts. The embryogenic pollen are small, starch-free with a clear cytoplasm whereas large starch-filled ones are nonembryogenic. The embryogenic pollen regularly form embryos at a frequency of 2% on a mineral medium supplemented with glutamine, asparagine and sucrose at pH 6.5.These results demonstrate, for the first time, that it is possible to have embryos in appreciable frequencies in ab initio pollen cultures raised from cold treated anthers.On leave from University of Delhi.     

Induction of embryos in pollen cultures of Nicotiana sylvestris

Induction of embryos in pollen cultures was possible when young buds from plants induced to flower at 15°C were given a cold treatment at 10°C for 15 days. Following selective isolation of embryogenic pollen the cultures raised were given cold treatment at 10°C for 12 or more days. In such cultures the embryos appeared at a frequency of 1.2% of the cultured pollen on mineral-sucrose medium at pH 6.8.     

Androgenesis in isolated pollen cultures ofNicotiana tabacum: Dependence upon pollen development

Summary The influence of the stage of pollen development and of the growth conditions of donor plants on the performance of cultures of isolated pollen fromNicotiana tabacum, var. Badischer Burley has been studied. The method described includes cold treatment (4–5 °C for 3 days) and a pre-culture of the anthers for 7 days at 24 °C before the pollen is isolated. With this system reproducible results were obtained with pollen at the early binucleate stage collected from plants 11–13 weeks old. Another prerequisite for reproducibility is that the donor plants must have been grown for eight weeks in soil with an additional supply of mineral salts. Furthermore, the production of haploids by these pollen cultures was strongly influenced by the photoperiodic and temperature regime experienced by the donor plants; it was best (0.07%) with pollen from short-day plants (8 hours light per day at 18 °C) and rather weak (0.015%) with pollen from long-day plants (16 hours light per day at 24 °C). In contrast to other reports, haploid production from anther cultures was not influenced by the photoperiod or temperature.Cytological studies undertaken at the end of the pre-culture period showed that there were no differences in the percentage of potential embryos for the stages of the late uninucleate, 1. pollen mitosis and early binucleate pollen of long-day plants (1.5%). This value was considerably higher with pollen from short-day plants (7–9%), indicating that short-day conditions at 18 °C of the donor plants are favourable for the induction of androgenesis. However, only the potential embryos formed by the pollen at the initial binucleate stage were able to continue androgenetic development after isolation.     

Environmental control and evidence for predetermination of pollen embryogenesis inNicotiana tabacum pollen

Summary The effect of daylenght and temperature for the donor plants (Nicotiana tabacum var. Badischer Burley) on the formation of pollen competent for embryogenesis (P-pollen) by the three possible routes (during normal flower developmentin situ (pollen dimorphism), during cold-treatment of excised flower buds, in cultured anthers) was studied. In all three routes, P-pollen frequency (premitotic pollen, before 1. sporophytic division, PPF) was affected in essentially the same way. At 24 °C and long days, PPF was low and short days had only a slightly increasing effect. At 18 °C and long days, PPF was higher and short days further increased it. Correlated with PPF under the different growth regimes was the percentage of units with more than one vegetative-type nucleus (normal embryos + abortive embryos + multinucleate pollen) in 3 weeks old anther cultures. Under greenhouse conditions, PPF was generally higher than at 24° in growth rooms and showed a maximum in the winter months. Plant age did not affect PPF. These results give further evidence that pollen embryogenesis is predetermined before excision and culture of the pollen or anthers.     

In vitro pollen embryogenesis in Nicotiana tabacum L. and its relation to pollen sterility,sex balance,and floral induction of the pollen donor plants

Pollen sterility, sex balance, and floral induction of the pollen donor plants were tested for a possible relation to embryogenesis from in vitro cultured tobacco pollen (Nicotiana tabacum L. var. Badischer Burley). The pollen grains destined to become embryos in culture (P-grains) were sterile for the donor plants as judged by their staining reaction with acetocarmine and fluorescin-diacetate, and by an in vitro germination test. They were produced in high frequency in flowers which exhibited a shift in sex balance towards femaleness. Sex balance could be measured by the relative length of pistil to stamens. High P-grain frequency, high pollen sterility, and a shift in sex balance towards femaleness could be induced by raising the donor plants under short days and/or low temperature (18–15° C) as compared to long days at 24° C. Short days and/or low temperature also reinforced floral induction, revealing that the tobacco variety Badischer Burley is a quantitative short day and low temperature plant and that the variety follows the rule that conditions of strong floral induction shift sex balance towards femaleness. At 12° C and short days, contabescent flowers were formed with completely sterile anthers containing a few and mostly collapsed P-grains. Based on these results, it is now possible to predict conditions by which haploids via pollen embryogenesis might be produced in high frequency from low-yielding and recalcitrant species.Abbreviations DPF dead pollen grain frequency - LD24 long days at 24° C - PD pollen dimorphism - P:S ratio of pistil to stamen length - SD15 short days at 15° C     

Embryogenesis and plant regeneration from isolated microspores of Brassica rapa L. ssp. Oleifera

Summary Conditions favourable to embryogenesis from isolated microspores of Brassica rapa L. ssp. oleifera (canola quality) were identified. A population with enhanced responsiveness for microspore embryogenesis (C200) was synthesized by crossing individual plants showing microspore embryogenic potential. For optimal microspore embryogenesis, buds (2–3mm in length, containing mid-late uninucieate microspores) were collected from older plants (2 months old) and microspores isolated and washed in iron-free B5 medium. NLN medium with its iron content reduced to half was beneficial for initial microspore culture. An elevated temperature(33–35°C) during the first day of culture, followed by maintenance at 25°C resulted in dozens of embryos from each isolation (about 100 buds). Seeds were obtained from plants regenerated from microsporederived embryos after colchicine treatment.     

Isolated pollen culture: plant reproductive development in a nutshell

Abstract

Isolated pollen can develop into two different directions when cultured in vitro. In a rich medium, microspores and young pollen grains develop into mature pollen that is fertile. When pollen is treated by a stress treatment such as a hunger treatment in a sucrose - and/or nitrogen - free medium, embryogenic pollen is formed that, after transfer to a rich medium, develops into embryos and haploid plants. This system of isolated pollen culture offers an opportunity to study two developmental processes, i.e. pollen development and embryogenesis, as well as a basic regulatory event, i.e. the transition from the gametophytic to the sporophytic phase in the alternation of generations in higher plants. In addition, both systems offer various application-oriented possibilities, such as production of doubled haploids, to overcome self-incompatibility, to rescue sterile pollen, pollen selection and pollen transformation. An understanding of the cell biological and molecular events during embryogenic induction may promote a wider application of doubled haploid breeding and the use of such plants for gene transfer.     

Pollen dimorphism in relation to pollen plant formation

Induction of haploid plants from pollen grains on culture of anthers has been possible in a number of angiosperms but it is not yet understood why only a fraction of pollen responds to form haploids. Noteworthy in this connection are the recent experiments on anther culture of barley, tobacco and wheat, where it has been pointed out that pollen populations are basically dimorphic. Pollen capable of forming haploids occur in a low frequency, arc smaller, and different from the majority of pollen destined to form gametes. Particularly in tobacco it has been possible to increase the frequency of pollen capable of forming embryos by subjecting plants to low temperature prior to flowering, and to achieve differentiation of embryogenic pollen by subjecting young buds from such plants to a prolonged cold treatment. On selective isolation, from the rest of the pollen, the embryogenic pollen from such buds readily form embryos at high frequency on a simple mineral-sucrose medium. The embryogenic pollen are repressed for gametophytic differentiation and in culture they differentiate to produce embryos. These experiments provide evidence that only certain pollen are capable of forming haploids.     

Nuclear DNA synthesis during the induction of embryogenesis in cultured microspores and pollen of Brassica napus L.

The dynamics of nuclear DNA synthesis were analysed in isolated microspores and pollen of Brassica napus that were induced to form embryos. DNA synthesis was visualized by the immunocytochemical labelling of incorporated Bromodeoxyuridine (BrdU), applied continuously or as a pulse during the first 24 h of culture under embryogenic (32 °C) and non-embryogenic (18 °C) conditions. Total DNA content of the nuclei was determined by microspectrophotometry. At the moment of isolation, microspore nuclei and nuclei of generative cells were at the G1, S or G2 phase. Vegetative nuclei of pollen were always in G1 at the onset of culture. When microspores were cultured at 18 °C, they followed the normal gametophytic development; when cultured at 32 °C, they divided symmetrically and became embryogenic or continued gametophytic development. Because the two nuclei of the symmetrically divided microspores were either both labelled with BrdU or not labelled at all, we concluded that microspores are inducible to form embryos from the G1 until the G2 phase. When bicellular pollen were cultured at 18 °C, they exhibited labelling exclusively in generative nuclei. This is comparable to the gametophytic development that occurs in vivo. Early bicellular pollen cultured at 32 °C, however, also exhibited replication in vegetative nuclei. The majority of vegetative nuclei re-entered the cell cycle after 12 h of culture. Replication in the vegetative cells preceded division of the vegetative cell, a prerequisite for pollen-derived embryogenesis.     

Pollen from cold-treated Nicotiana tabacum buds: Embryogenic capacity,peroxidase activity and partitioning in aqueous two-phase systems

Pollen isolated from fresh and cold treated tobacco (Nicotiana tabacum L. ew. Wisconsin 38) flower buds were separated using aqueous polymer two-phase partitioning and analysed with respect to embryogenic capacity, peroxidase activity and isoperoxidase pattern. Pollen with embryogenic capacity from cold-treated flower buds were enriched in fractions with higher partitioning than those from fresh flower buds, but the amounts were the same. Cold led to a general increase in specific peroxidase activity in pollen fractions enriched in embryogenic pollen, and also to specific changes in the isoperoxidase pattern. Neutral peroxidase species (pI around 7) and alkaline species (pIs around 9) could be related to pollen fractions enriched in embryogenic pollen. The data agree with earlier data showing that the amount of pollen with the potential to form embryos is established at an early stage in flower development, whereas if they really do so depends on how they are pretreated, e.g. by cold treatments of the buds. The latter is also reflected by quantitative and qualitative differences in peroxidase.     

Plant transformation by particle bombardment of embryogenic pollen

Summary Direct delivery of DNA into embryogenic pollen was used to produce transgenic plants in tobacco. A plasmid bearing the ß-glucuronidase (GUS) marker gene in fusion with the 35S-promoter was introduced by microprojectile bombardment into mid-binucleate pollen of Nicotiana tabacum that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 10⁴ pollen grains were GUS⁺. Visual selection by staining with a non-lethal substrate for GUS was used to manually isolate transformed embryos. From the initial population of embryogenic GUS⁺ pollen, 1–5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transformants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and backcrosses of one transformant were tested for GUS expression and by Southern blots. All F1-plants were transgenic, in accordance with Mendelian inheritance.Abbreviations GUS ß-glucuronidase - CaMV Cauliflower Mosaic Virus - MCS multicellular structure - NPTII neomycin phosphotransferase - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide - DAPI 4,6-diamidino-2-phenylindole - Tris Tris(hydroxymethyl)aminomethane hydrochloride - EDTA ethylenedinitrilo tetraacetic acid, disodium salt dihydrate     

High frequency production of embryos inDatura innoxia from isolated pollen grains by combined cold treatment and serial culture of anthers in liquid medium

Summary This study concerns the development of pollen embryos as affected by various physical conditions of culture in media devoid of hormones. Freshly isolated pollen, from anthers ofDatura, failed to form embryos regardless of whether they were cultured on liquid or solid medium. In contrast, pollen isolated from anthers precultured on solid medium did form embryos and the response could be increased by prior cold treatment of anthers at 4 °C for 4 days. However, the best results were obtained when anthers were cultured from the very beginning in liquid medium and transferred serially to fresh medium. Under such conditions, the anthers dehisced, allowing spontaneous shedding of pollen grains. It was thus possible to have several fractions of shed pollen continuing their development into embryos. When serial culture was started with anthers from cold-treated buds not only were embryos formed in all the fractions of shed pollen but the frequency was also considerably higher than in any mode of culturing.     

Stress as the major signal controlling the developmental fate of tobacco microspores: towards a unified model of induction of microspore/pollen embryogenesis

Specific stress treatments (sucrose starvation, alone or combined with a heat shock) applied to isolated tobacco (Nicotiana tabacum L.) microspores irreversibly blocked normal gametophytic development and induced the formation of embryogenic cells, which developed subsequently into pollen-derived embryos by culture at 25°C in a sugar-containing medium. A cold shock at 4°C did not inhibit microspore maturation in vitro and did not induce cell division activity, even when combined with a starvation treatment. In the absence of sucrose, microspores isolated in the G1 phase of the cell cycle replicated their DNA and accumulated in G2. Late microspores underwent miotosis during the first day of culture which resulted in a mixed population of bicellular pollen grains and uninucleate microspores, both embryogenic. After the inductive stress treatments the origin of the first multicellular structures, formed in the sugar-containing medium, could be traced to divisions of the microspore cell or divisions of the vegetative cell of bicellular pollen, indicating that the symmetry of microspore mitosis in vitro is not important for embryogenic induction. These results represent a step forward towards a unified model of induction of embryogenesis from microspores/pollen which, within a relatively wide developmental window, are competent to deviate from normal gametophytic development and initiate the alternative sporophytic programme, in response to specific stress signals.Abbreviation DAPI 4,6-diamidino-2-phenylindole We acknowledge the help of Monica Boscaiu and Zarko Hrzenjak with the artwork, and Michaela Braun-Mayer for growing the tobacco plants. This project was financed by the Austrian Fonds zur Forderung der wissenschaftlichen Forschung, grant S6003-BIO.     

Separation of precultured pollen from Nicotiana tabacum by aqueous polymer two-phase partition

Pollen isolated from cold treated and precultivated anthers of tobacco (Nicotiana tabacum L. var. Wisconsin 38) were separated into different fractions with counter-current distribution using an aqueous Dextran-polyethylene glycol two-phase system. It was possible to distinguish among eight pollen classes differing in developmental stage and in partitioning. A part of each fraction was cultivated for analysis of embryo formation. This was highest in a fraction with an intermediate to high partition in the phase system. Enriched in this fraction were also pollen that were fairly well stained with acetocarmine, contained several nuclei and had a relatively low mitochondrial activity. The enrichment of embryogenic pollen offers several advantages especially to physiological studies on embryogenesis.     

Stress-induced microspore embryogenesis in tobacco: an optimized system for molecular studies

Summary Specific stress treatments applied to isolated tobacco (Nicotiana tabacum L.) microspores efficiently induced haploid embryo formation in vitro. A heat shock at 33 or 37°C in the presence of sugar, as well as sucrose-starvation at 25°C, resulted in the formation of embryogenic microspores. A combination of both treatments had an additive effect. Under optimal induction conditions all viable microspores in the culture were embryogenic and developed subsequently into pollen embryos by culture at 25°C in a sugar-containing medium, with induction frequencies of more than 70% with respect to the initial microspore population. A high fraction of the early pollen embryos continued their development in vitro, giving rise to haploid plants. In contrast to other available systems for microspore/pollen embryogenesis, the new protocol allows the production of homogeneous populations of embryogenic microspores and early globular embryos in large-scale cultures, without any purification step, and is therefore well suited for biochemical and molecular work.Abbreviations EDTA ethylenediaminetetraacetate - DAPI 4,6-diamidino-2-phenylindole     

Recovery and regeneration of embryogenic cultures from female flowers of False Horn Plantain

Somatic embryogenesis from immature male flowers in Musa is only suitable for genotypes with a male bud. Six friable embryogenic cultures were obtained from 28 cultured buds of female flowers of the AAB False Horn Plantains, ‘Curraré’ and ‘Curraré Enano’. Embryogenic suspensions were established from these embryogenic cultures. Somatic embryogenesis was demonstrated histologicaly. Regeneration of plants was obtained either from somatic embryos directly isolated from embryogenic cultures or from suspensions after plating on a semi-solid medium. This study demonstrates that somatic embryogenesis from immature flowers is suitable for genotypes of Musa with or without male buds. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of photoperiod and temperature on sexual recrudescence in the male weakfish,Cynoscion regalis

Synopsis Male weakfish,Cynoscion regalis, were collected in the south-west portion of Delaware Bay from April through August of 1992. Histological examination of testes collected from these specimens was used as a baseline for comparison with laboratory data. Weakfish testes were found to be of the unrestricted, continuous spermatogenic type and spermatogenesis was apparently prenuptial. The effects of photoperiod and temperature on gonadal maturation were studied in the laboratory using fish collected in June and July 1991. These fish were exposed to two months of simulated winter conditions prior to assignment to one of four experimental photoperiod/temperature regimes. The treatments included combinations of long day (LD,15 h light) or short day (SD, 9 h light) and high (HT, 20° C) or low (LT, 13° C) water temperatures. The four treatment groups were LD/HT, LD/LT, SD/HT, SD/LT. The LD/HT group was the only one to mature fully, expressing increased plasma androgen levels, increased gonadosomatic index (GSI) and advanced spermatogenesis, as well as hypertrophy of the sonic muscles, a seasonally expressed secondary sexual characteristic of male weakfish. High temperatures promoted the later stages of spermatogenesis, which were apparently inhibited by low temperatures. The presence of an endogenous annual cycle is suggested by the partial maturation of the testes and sonic muscles in all treatment groups, regardless of photoperiod/temperature regime.