Regulation of flower development in cultured ears of maize (Zea mays L.)

Summary Young ears of maize were cultured in two different liquid media containing either kinetin (KN) or kinetin + gibberellic acid (KN + GA₃) in order to manipulate stamen and gynoecium development. In KN medium, stamens developed and gynoecia aborted in the flowers of the cultured immature ears. In the KN + GA₃ medium, however, ovaries with silks developed and stamens aborted. These differential morphological events were recorded with SEM photomicrographs at regular intervals after excision of ear inflorescences. In addition, the mitotic activity in the developing or aborting organs was determined over a 75-h period. It increased from 6% to 14% in developing organs (i.e. stamens in KN medium, and gynoecia in KN + GA₃ medium) and gradually decreased to 1% in the degenerating organs (i.e. gynoecia in KN medium, and stamens in KN + GA₃ medium) by 45 h of culture. The mitotic activity reached zero in degenerating flower organs by 75 h of culture. Whether these differential sensitivities to the exogenously applied members of these two plant growth regulator classes are unique to our in vitro system or reflect a more general control feature of in vivo inflorescences must await further clarification.     

Maturation of maize pollen in vitro

Summary Maturation of maize pollen was obtained in male reproductive structures cultured in vitro. Immature tassels containing microspores at the mid-uninucleate to late-binucleate stage of development were excised and spikelets, anthers, and/or isolated microspores were cultured on a medium capable of supporting pollen maturation. Microspore mitosis, culminating in the production of starch-filled, trinucleate pollen capable of germination, was observed after 7–15 days, depending on the genotype and stage at which the cultures were initiated. Up to 100%, 70%, and 20% of the cultured spikelets, anthers, and isolated microspores, respectively, produced mature pollen, which germinated, however, at different frequencies (i.e., spikelets, 50–70%; anthers, 5–10%; microspores, <1%). Mature kernels were produced following fertilization with pollen from cultured spikelets and anthers. These procedures provide methods for the in vitro manipulation of a significant phase of the maize life cycle.     

IN VITRO CULTURE OF EAR SHOOTS OF ZEA MAYS AND THE EFFECT OF KINETIN ON SEX EXPRESSION

Immature ear primordia of maize when cultured on a defined liquid medium grew and differentiated in response to two variables: 1) size of initial explants and 2) concentration of kinetin in the medium. Overall growth of primordia, less than 15 mm at explanting, reached an optimum fresh weight and ear length when kinetin was 10^–6m. Ears less than 10 mm, in the presence of kinetin, produced many male spikelets with well-developed stamens in both upper and lower flowers. Ears greater than 15 mm, at explanting, produced only female flowers regardless of the kinetin concentration. Different proportions of male and female and bisexual flowers were produced depending upon the initial size of inflorescences and the concentration of kinetin.     

Production of doubled haploid rice plants (Oryza sativa L.) by anther culture

The results of anther culture of F₂ pollen issued from 23 single crosses are presented. A relation between the morphology of the panicle and the microspore stage was established. After cold-pretreatment (8 days at 4°C), the anthers were cultured on the callus-induction medium N6 supplemented with 1 mg l^–1 naphthaleneacetic acid. The calli were transferred to MS plant regeneration medium supplemented with 3 mg l^–1 kinetin + 0.5 mg l^–1 naphthaleneacetic acid. The induction frequency varied from 0.22% to 29% and the regeneration frequency from 0% to 144.4%, dependent upon the crosses used. On average, 27% of the plants obtained were albinos and 59% of the green plants underwent spontaneous chromosome doubling. Thirtynine doubled haploid lines were evaluated and multiplied in the field. Lines with an excellent behaviour in upland culture conditions were selected from two crosses.     

Enhanced seed development in the interspecific cross Allium cepa x A. sphaerocephalon through ovary culture

Allium sphaerocephalon pollen tubes grew into styles and penetrated micropyles of Allium cepa, but ovules started to degenerate about 16 days after pollination and no seeds developed. Seeds developed in vitro in ovaries excised from flowers 4 and 7 days after pollination. Seven weeks after culture initiation, seeds had grown in 4 of 96 excised ovaries, cultured on BDS medium supplemented with GA₃. Although the culture medium supported seed maturation within excised ovaries of self-pollinated A. cepa flowers, no viable hybrid seeds were recovered from crosses with A. sphaerocephalon. Extended post-fertilization barriers may have restrained development of hybrid embryos in vitro. Ovary culture followed by in ovulo embryo rescue may be feasible for distant-species hybridization in Allium.     

In vitro culture of immature tassels of an inbred field variety of Zea mays,cv. Oh43

The medium and conditions which permit in vitro culture of immature tassels of Zea mays cv. Oh43 are reported. Final fresh weight, total number of spikelets and the number of normal spikelets were enhanced by optimal concentrations of sucrose (0.3 M), kinetin (10^–7 M) and casein hydrolysate (30 mg l^–1) when added to the Murashige and Skoog salts, White's vitamins and glycine and inositol. A single cultured tassel produces up to 200 normal spikelets which contain anthers bearing germinable pollen.     

Fertilization and seed production with pollen from in-vitro-cultured maize tassels

Immature tassel meristems (1–1.5 cm) of maize (Zea mays L.) explanted to a defined nutrient medium underwent further growth and floral development. Microsporogenesis, gametogenesis and pollen maturation were completed within 25 d in vitro. The pollen, recovered from the cultured tassel, germinated on nutrient agar and also on receptive silks. Viable seed produced from controlled pollinations germinated and grew into mature, normal plants. Thus, a significant component of the life cycle of maize can be completed in vitro where analyses and manipulations are possible for both basic and applied research.     

Pollination between maize and teosinte: an important determinant of gene flow in Mexico

Gene flow between maize [Zea mays (L.)] and its wild relatives does occur, but at very low frequencies. Experiments were undertaken in Tapachula, Nayarit, Mexico to investigate gene flow between a hybrid maize, landraces of maize and teosinte (Z. mays ssp. mexicana, races Chalco and Central Plateau). Hybridization, flowering synchrony, pollen size and longevity, silk elongation rates, silk and trichome lengths and tassel diameter and morphology were measured. Hybrid and open-pollinated maize ears produced a mean of 8 and 11 seeds per ear, respectively, when hand-pollinated with teosinte pollen, which is approximately 1–2% of the ovules normally produced on a hybrid maize ear. Teosinte ears produced a mean of 0.2–0.3 seeds per ear when pollinated with maize pollen, which is more than one-fold fewer seeds than produced on a maize ear pollinated with teosinte pollen. The pollination rate on a per plant basis was similar in the context of a maize plant with 400–500 seeds and a teosinte plant with 30–40 inflorescences and 9–12 fruitcases per inflorescence. A number of other factors also influenced gene-flow direction: (1) between 90% and 95% of the fruitcases produced on teosinte that was fertilized by maize pollen were sterile; (2) teosinte collections were made in an area where incompatibility systems that limit fertilization are present; (3) silk longevity was much shorter for teosinte than for maize (approx. 4 days vs. approx. 11 days); (4) teosinte produced more pollen on a per plant basis than the landraces and commercial hybrid maize; (5) teosinte frequently produced lateral branches with silks close to a terminal tassel producing pollen. Collectively these factors tend to favor crossing in the direction of teosinte to maize. Our results support the hypothesis that gene flow and the subsequent introgression of maize genes into teosinte populations most probably results from crosses where teosinte first pollinates maize. The resultant hybrids then backcross with teosinte to introgress the maize genes into the teosinte genome. This approach would slow introgression and may help explain why teosinte continues to co-exist as a separate entity even though it normally grows in the vicinity of much larger populations of maize.     

In vitro regeneration of Camellia sinensis (L.) O. Kuntze by somatic embryogenesis

Within 3 weeks of culture, excised cotyledon expiants of Camellia sinensis (L.) O. Kuntze produced somatic embryos without intermediate callus when cultured in Murashige and Skoog's basal medium with 30 g^–1 sucrose. In medium without plant growth regulators, up to 60% of the cultures developed somatic embryos. Embryogenic competence was reduced by increasing concentrations of plant growth regulators tested (i.e. kinetin, 6-benzylaminopurine, and indole butyric acid). The somatic embryos developed, grew to maturity without being subcultured within 6–8 weeks. Secondary embryogenesis was not observed. Germination of isolated mature somatic embryos was low in medium without plant growth regulators. Up to 53% and 60% germination occurred when medium impregnated with kinetin at 1.8 mgl^–1 or 1.0 mgl^–1 6-benzylaminopurine were used respectively. Callus was also routinely produced when cotyledons were cultured in MS basal medium with auxins (2,4-dichlorophenoxyacetic acid and indole acetic acid). Callus induction was however, also achieved in plant growth regulator free medium. Indirect somatic embryogenesis was not induced in the present study.Abbreviations K kinetin - BAP 6-benzylaminopurine - IBA indole butyric acid - IAA indole acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA Naphthalene acetic acid - Fe-EDTA Ethylenediaminetetra-acetic acid (Ferric monosodium salt)     

Development of secondary inflorescences and in vitro plantlets from inflorescence cultures of Amaranthus paniculatus

Immature inflorescences of Amaranthus paniculatus were used as explants for in vitro culture studies. When placed on a medium supplemented with 3–6 mg/l kinetin, explants developed into secondary inflorescences. Leaves and shoots developed following culture of inflorescence tissue on media containing 8–15 mg/l kinetin or 5–10 mg/l BAP. These shoots when subcultured on MS medium supplemented with 12 mg/l kinetin + 15% coconut milk, formed roots. These rooted plantlets later flowered in vitro.Abbreviations MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - BAP 6-benzylaminopurine - Kn 6-furfurylaminopurine - CM coconut milk     

Induction of division and differentiation of somatic embryos in the leaf epidermis of Gaillardia picta

Somatic embroys and subsequent plant regeneration were obtained from isolated leaf epidermis of Gaillardia picta. Abaxial and adaxial epidermal peels (monolayer) from 45 days old aseptic seedlings were isolated and segments measuring 5 mm x 3 mm were cultured on B₅ basal medium supplemented with various growth regulators such as naphthaleneacetic acid or indolebutyric acid and benzylaminopurine or kinetin. Within 12 h of culture the epidermal cells showed receding of cytoplasm from the walls. After 48 h of incubation 3 or 4 localized zones, each consisting of 3–8 cells that accumulated cytoplasm and stained densely, were observed. Mitotic divisions occurred in these zones on day 3 of culture and localized masses of callus were observed in 95% of the cultures after 10 days. In another 5 days, the callus differentiated somatic embryos or roots, depending on the growth regulators and their concentration in the medium.Abbreviations Used BAP 6-benzylamiopurine - IBA indolebutyric acid - Kn kinetin - NAA -naphthaleneacetic acid     

High frequency androgenesis from isolated microspores of maize

Anthers from a highly androgenic genotype of maize (139/39-02), when cultured in a modified, liquid YP medium, dehisced within 2–7 days resulting in a stationary suspension of microspores. After 12–15 days, the microspore suspension was found to contain multicellular masses which went on to produce macroscopic embryo-like structures within 20–25 days of culture initiation. Embryogenic callus could be obtained by transferring microspore-derived embryos onto a modified N6 medium supplemented with 2.5 mg/l dicamba and 0.1 mg/l 2,4-D. Subculture onto hormone-free medium resulted in plant regeneration. Over 400 embryo-like structures per 100 anthers cultured have been obtained from liquid induction medium as compared to 55 embryos per 100 anthers cultured on an agar-solidified medium. Approximately 5–25% of these embryo-like structures went on to produce callus from which plants could be recovered. Mechanical isolation of microspores from anthers precultured for 0, 3, and 7 days also resulted in embryo production and plant regeneration. This represents the first report of plant recovery from isolated maize microspores. The use of a liquid induction medium applied to a highly androgenic genotype allows for the production of large numbers of microspore-derived plants and provides a single, haploid cell regeneration system for maize.     

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     

Nucleic acid and protein synthesis in discs cut from mature leaves of Nicotiana tabacum L. and cultured on nutrient agar with and without kinetin

The effect of kinetin on aspects of the metabolism of discs cut from mature leaves of Nicotiana tabacum and cultured in the light on agar containing mineral salts and sucrose was studied. In the first few days of culture there was a rapid decline in chlorophyll content. Discs treated with kinetin in the light began to resynthesise chlorophyll after 3–4 days and this was correlated with chloroplast replication. Kinetin promoted chloroplast replication but was not always essential. An increase in fresh weight also occurred, due mainly to cell expansion. Nitrate reductase activity increased rapidly during the first few hours after placing discs on the culture medium but kinetin had no effect on this reponse. Subsequently there were dramatic increases in RNA and protein content which were largely independent of kinetin. Gel electrophoresis showed that cytoplasmic and chloroplast ribosomal RNA and a large amount of soluble RNA were synthesised during culture of the discs. These results are discussed in relation to the role of kinetin in delaying leaf sensescence.     

Effects of activated charcoal, explant size, explant position and sucrose concentration on plant and shoot regeneration of Lilium longiflorum via young stem culture

An efficient system for the in vitro plant and shootregeneration of Lilium longiflorum was developed andaccomplished using transverse thin cell layers (tTCL) of young stems.tTCLs were cut transversely along young stems from which the shoot-tipshad been removed. Sections were measured accurately using a graded gridand were cut in 4 mm × 4 mm × 1 mm cubes, eliminatingepidermal tissue, and were cultured on one-half MS medium containing 8 gl^–1 agar, different sucrose concentrations (10, 20, 30 or 40g l^–1), and with or without 1 mg l^–1 activatedcharcoal (AC). Plants formed on the surface of tTCLs within 60 days onone-half MS medium containing 8 g l^–1 agar and 20 gl^–1 sucrose. Sections of 1 mm taken just below the apicalarea developed buds within 15 days, whereas the sections closer to thebase required about 45 days. Shoot regeneration was enhanced whensucrose concentration was used at 30 or 40 g l^–1 after 60days of culture. No root formation occurred. Both shooting and rootingoccurred when sucrose was used at 20 g l^–1. The plantletswere transferred to soil and grew well under greenhouseconditions.     

Organogenesis and embryogenesis inHelianthus tuberosus and in the interspecific hybridHelianthus annuus ×Helianthus tuberosus

A method of plant regeneration from cotyledons ofHelianthus tuberosus, Helianthus annuus ×Helianthus tuberosus and for the backcross of the interspecific hybrids onH. annuus was developed. Induction of somatic embryogenesis and plantlet regeneration from anther culture of the interspecific hybridsH. annuus ×H. tuberosus is reported.Cotyledons were cultured on Murashige and Skoog basal medium (MS) supplemented with indole-3-acetic acid (IAA) and 6-furfurylaminopurine (kinetin) or N⁶-benzylaminopurine (BAP). Shoot regeneration occurred on most of the media tested, but the best results were obtained on media with a high concentration of cytokinins (BAP or kinetin: 4 mg l^–1) and lower concentration of auxin (IAA: 0.5–1 mg l^–1).Embryogenic callus and adventitious buds were initiated from only two anthers of the hybridH. annuus ×H. tuberosus cultured on the MS medium containing BAP (0.2 mg l^–1) and 1-naphtalenacetic acid (NAA: 0.1 mg l^–1). Prolonged culture of these embryogenic calli and buds on the original medium with successive subculture on MS basal medium without growth regulators resulted in embryo formation and shoot differentiation. The plantlets, after rooting, were established in soil.     

Studies on Conditions for Cell Division and Embryogenesis in Isolated Pollen Culture of Nicotiana rustica

A method for the induction of a high rate of cell division and embryogenesis of Nicotiana rustica pollen was developed. Binucleate pollen grains were fractionated by Percoll density gradient (35/45%) centrifugation and cultured in 0.4 molar mannitol at 30°C (the first culture). After 3 days in culture pollen was recollected by a second Percoll fractionation (0/30%) and transferred to and cultured in a medium containing the Murashige-Skoog macro-elements, 0.4 molar mannitol, 40 millimolar galactose, 3 millimolar glutamine, and 5 micromolar ABA for 10 days (the second culture). The cell population consisting of about 80% dividing pollen was transferred to a Murashige-Skoog medium containing 0.4 molar mannitol, 3 millimolar glutamine, and no phytohormone (the third culture), where about 40% of dividing pollen developed into embryos or embryogenic calli.     

Plant Regeneration from Protoplasts of Corn(Zea mays L.)

Maize embryogenic calli induced from pollen were subcultured for one and one half years on N, basic medium supplemented with 2 mg/1 kinetin, 1 mg/l 6-benzyl-aminopurine, 0.3 mg/l 2,4-D, 500 mg/l casein hydrolysate and 250 mg/l glutamine. These embryogenic calli were used for protoplast isolation. Protoplasts were cultured on Z2 medium (Table 1) which is composed of rice protoplast culture basic medium 1 supplemented with 0.2 mg/l kinetin, 0.1 mg/l 6-benzyl-aminopurine, 0.5 mg/l 2,4-D, 200 mg/l casein hydrolysate, 100 mg/l glutamine and 2% coconut milk. The first division of regenerated cell occurred after 4-6 days in culture. After 3 weeks later, small calli could be seen with naked eyes. At this moment, addition of the same Z2 medium with decreased osmoticum twice for the protoplast culture is necessary. Regenerated calli, 2–4 mm in diameter, were transferred in succession on differentiation medium Z3 and Z4 for organogenesis. Embryogenesis and plant regeneration could occur simultaneously on Z4 differentiation medium. It seems that except the cultural conditions genotype and using of embryogenic materials are the two key factors for plant regeneration of maize protoplast and the former may be the critical one.     

Fiber development in preanthesis cotton ovules

A tissue culture method was developed to investigate the production of cotton (Gossypium hirsutum L. cv. Texas Marker-1) fibers in vitro. Ovules were excised from 3, 5, 7 and 9 days preanthesis ovaries and placed on an agar-solidified, modified Murashige and Skoog medium containing 2.3 μ M kinetin and 0.45 μ M –2,4–dichlo-rophenoxyacetic acid or 2.3 μ M kinetin and 10.7 μ M naphthaleneacetic acid. Ovules formed fibers and callus tissue. Fibers formed in vitro were up to 10 mm long, 10–22 μ wide and the cell wall was 1–3 μ M thick. Callus tissue cells were subcultured for over 25 weeks and their degree of elongation was monitored. The ability of ovule-derived cells to direct expansion in a longitudinal direction diminished, while lateral expansion increased with time in culture.     

in Vitro Somatic Embryogenesis in Typhonium Trilobatum Schott.

An in vitro protocol was developed for the production of plants via somatic embryogenesis in callus cultures derived from petiole and leaf explants of Typhonium trilobatum. Optimum callus formation was achieved on semisolid Murashige and Skoog's [9] medium supplemented with 0.25 mg L–1 kinetin and 3.0 mgL–1 1-naphthaleneacetic acid (NAA) after 6 weeks of culture. Somatic embryogenesis was achieved upon transferring the callus to a medium containing 1.0 mg Lminus 1 kinetin and 0.25 mg Lminus 1 NAA. In vitro tuberization was also achieved on medium containing 1/2 strength MS basal salts supplemented with 1.0 mg L–1 Kinetin and 0.1 mg L–1 NAA. Embryo maturation and germination was achieved on the MS basal salts supplemented with 0.01 mg L–1 NAA and 2% (w/v) sucrose. Some thousands somatic embryo derived plantlets were hardened in the greenhouse and eventually planted in the open field.