WO1991002052A1 - Asssay for molecules affecting plant morphogenesis - Google Patents

Abstract

A plant thin-cell-layer (TCL) culture system has been developed to study root, vegetative shoot, and flower morphogenesis. The effects of hormone concentrations, pH, and light quality and quantity on TCL morphogenesis were analyzed. Roots, vegetative shoots, and flowers formed on TCLs cultured on various liquid media that had been adjusted to the same pH. Evidence is presented showing that the effects of light on TCL morphogenesis are associated with light-mediated degradation of indol-3-butyric acid (IBA) in the medium. Evaluation of the organogenesis that occurred in TCLs cultured on a range of IBA and kinetin concentrations revealed that the quantity and type of organs formed were dependent upon the concentration of IBA and kinetin in the medium. The TCL assay system is useful for studies of the molecular changes associated with organogenesis and for the screening, identification, and purification compounds capable of affecting plant morphogenesis.

Description

*-/ - ASSAY FOR MOLECULES AFFECTING PLANT MORPHOGENESIS

Background of the Invention

The U.S. Government has certain rights in this invention by virtue of NIH grant no. F32 GM11857-02, DOE grant no. DE-FG09- 85ER13425, and in part by Department of Energy grant no. DE-FG09-8713810 as part of the USDA/DOE/NSF Plant Science Centers Program.

Morphogenesis, the sum of processes that give form to an organism, includes organization of cells into tissues, tissues into organs, and organs into an entire organism (Wareing and Phillips, Growth and Differentiation in Plants. 21, 165-167, 323 (Pergamen

Press, Oxford 1970)). In plants, morphogenesis is a continual process. A growing axis that consists of two centers of division, the root and shoot meristems, is established in the embryo. The entire plant body, including roots, leaves, stems, and flowers, is formed from these meristematic centers.

Unlike animal development, plant development is notably plastic. Differentiated cells can become meristematic (undifferentiated and totipotent) during normal development or in response to wounding. The totipotency of plant cells is demonstrated by the ability of in vitro cultured plant organs, tissues, cells, and protoplasts to form meristematic centers that develop into roots, vegetative shoots, flowers, or entire plants.

Much progress has been made in identifying factors that regulate plant morphogenesis. Examples of such factors include environmental stimuli such as light and temperature (Sussex, Cell 56, 225-229 (1989)), as well as molecules endogenous to plants, such as the phytohormone cytokinin, auxin (Davies, Plant Hormones and Their Role in Growth and Development, ed. P . Davies, 1-11 (Martinus Nijhoff Publishers, Dordrecht 1987)). Other phytohormones include abscisic acid, ethylene, and gibberellin. Work by Skoog and Miller, Svmp. Soc. Exp. Biol. XI, 118-131 (1957), and Tran Thanh Van, et al. Planta 119, 149-159 (1974), has established that the concentrations of auxin and cytokinin in media can determine the type of organogenesis that occurs in callus and tissue cultures. Studies with auxin mutants (Estelle and Somerville, Mol. Gen. Genet. 206, 200-206 (1987)) and with plants transformed with T-DNA cytokinin (Smigocki and Owens, Proc. Natl. Acad. Sci. 85, 5131-5135 (1988); Memelink et al., Dev. Genet. 8, 321-337 (1987)) and auxin synthesis genes (Klee et al., Genes Dev. 1, 86-96 (1987)) have provided evidence that exogenous application of auxin and cytokinin affects organogenesis of tissue cultures in a manner similar to that which occurs in the intact plant It is likely, however, that the actual role of auxin and cytokinin concentrations in organogenesis will remain unresolved until the mechanisms by which hormones regulate growth and development are understood gt the molecular level.

Other factors affecting plant morphogenesis also need to be studied at the molecular level. In order to address the need to study morphogenic events at the molecular level, and to simplify plant tissue culture and to minimize cellular variables, thin-cell-layer (TCL) culturing techniques have been developed. Thin-cell-layer explants consist of approximately five to ten cytologically differentiated cells, the epidermal and subepidermal cells, in which most of the cell-to-cell contacts are maintained. Specifically, strips of tissue, approximately 1 mm wide consisting of one layer of epidermal cells, two to three layers of chlorenchyma and three to six layers of collenchyma and/or parenchyma, are cut from the floral branch tissue and used in TCL cultures.

Tobacco TCL's have been used recently to show that other molecules of plant origin, oligosaccharins, are capable of affecting morphogenesis (Gollin et al, Biol. Cell 51, 275-280 (1984); York et al., Plant Physiol. 72, 295-297 (1984); Tran Thanh Van et al, Nature 314, 615-617 (1985); McDougall and Fry, Planta 175, 412-416 (1988)). Thin-cell-layer explants have been used in a number of laboratories for developmental studies, particularly studies of the flowering process (Tran Thanh Van, et al, 1974; Bridgen and Veilleux, J. Amer. Soc. Hort. Sci. 110, 233-236 (1985); Altamura et al., Plant Science 46, 69-75 (1986); Wilms and Sassen, New Phvtol. 105, 57-65 (1987); Kaur- Sawhney et al., Planta 173, 282-284 (1988); Smulders et al, J. Exp. Bot, 39, 451-459 (1988)).

Most of these studies were done by incubating TCLs taken from the pedicel on agar-solidified media. TCLs have also been taken from the basal interaodes of primary floral branches and incubated on liquid media with defined concentrations of auxin and cytokinin and a determined pH in order to investigate the effect of cell wall fragments on the formation of roots, vegetative shoots, and flowers (Cousson and Tran Thanh Van, Physiol. Plant 51, 77-84 (1981); Tran Thanh Van, et al, 1985; Rajeevan and Lang, Planta 171, 560-564 (1987); Meeks- Wagner et al., The Plant Cell 1, 25-35 (1989)). Tran Thanh Van, et al., presented evidence that the addition of plant cell wall fragments to TCLs cultured on various liquid media caused TCLs to form vegetative shoots rather than flowers or callus, and flowers rather than vegetative shoots.

However, in spite of extensive efforts to perform the in vitro TCL assay as described by Tran Thanh Van, et al., (1985), it has not been possible to consistently repeat the published results and obtain expected and reproducible TCL organogenesis.

It is therefore an object of the present invention to provide a simple, defined, reliable, and reproducible TCL system for the study of plant morphogenesis.

It is a further object of the present invention to use such a reproducible TCL system to screen for and assay bioactive molecules capable of affecting plant morphogenesis.

It is a still further object of the present invention to use the TCL assay system to aid in the purification of bioactive molecules able to affect plant morphogenesis. It is yet a further object of the present invention to use the TCL system to study plant morphogenesis at the molecular level.

Summary of the Invention

A plant thin-cell-layer (TCL) culture system has been developed to study root, vegetative shoot, and flower morphogenesis. The effects of hormone concentrations, pH, and light quality and quantity on TCL morphogenesis were analyzed. Roots, vegetative shoots, and flowers formed on TCLs cultured on various liquid media that had been adjusted to the same pH. The type and number of organs formed were sensitive to the intensity of light (55, 75, 100, and 120 _/iEinsteins m^"2sec-l) under which TCLs were grown. Evidence is presented showing that the effects of light on TCL morphogenesis are associated with light-mediated degradation of indol-3-butyric acid (IBA) in the medium. Evaluation of the organogenesis that occurred in TCLs cultured on a range of IBA and kinetin concentrations revealed that the quantity and type of organs formed were dependent upon the concentrations of IBA and kinetin in the medium.

The TCL assay system is useful for studies of the molecular changes associated with organogenesis as well as for the screening, identification, and purification compounds capable of affecting plant morphogenesis.

An example is provided demonstrating the usefulness of the assay in screening for molecules having an effect on morphogenesis, wherein specific molecules present in pectic cell wall fragments were shown to regulate morphogenesis.

Brief Description of the Drawings

Figure 1 illustrates the typical morphology of Nicotiana tabacum L. cv Samsun where T represents a terminal floral branch and 1-4 represent consecutive primary floral branches. Arrows denote floral branches used in the TCL assay. The total number of leaves varies from 16-29 and the total number of flower branches with mature flowers varies from 3-6.

Figure 2 is a graph showing the effect of pH on the number of flowers (F), vegetative shoots (VS), and roots (R) formed per TCL in TCLs cultured under 55 μEm^'V¹ of cool white light for 23-25 days on basal medium containing 0.5 μM kinetin and (A) 0.5 μM IBA, (B) 5 μM IBA, and (C) 15 μM IBA Data represent two experiments with a minimum of 24 replicates per experiment.

Figure 3 is a graph showing the effect of cool white light quantity on the mean numbers of flowers (F), vegetative shoots (VS), and roots (R) formed per Petri dish of 20 TCLs after 23-25 days in culture on basal medium containing (A) 0.5 μM IBA and 0.5 μM kinetin, pH 3.8; (B) 3 μM IBA and 0.5 μM kinetin, pH 6.15; and (C) 7 μM IBA and 0.2 μM kinetin, pH 5.8. Data represent one experiment with 7 or 8 replicate Petri dishes. Standard error bars were less than 0.5 mm in length.

Figure 4 is a graph of the effect of cool white light quantity on the mean numbers of flowers (F), vegetative shoots (VS), and roots (R) formed per TCL after 23-25 days in culture on basal medium, pH 5.8, containing (A) 0.5 μM IBA and 0.5 μM kinetin, (B) 4 μM IBA and 0.5 μM kinetin, (C) 15 μM IBA and 0.5 μM kinetin, and (D) 1 μM IBA and 6 μM kinetin. Data represent two experiments with 10-48 replicates per experiment. Standard error bars were less than 0.5 mm in length. Figure 5 is a graph showing the effect of cool white light quantity on the stability of IBA in the medium. Mean concentration (± SE) of IBA remaining in the medium after exposure of medium containing 6 μM IBA and 0.5 μM kinetin to 55 (55L) or 120 (120L) μEπf ²sec^{" 1} of cool white fluorescent light for the indicated length of time. The media not exposed to light were also tested (55D and

120D). Data represent duplicate samples from one (55 μEπTV¹ and 12 day point from 120 μEπfV¹) or two (120 μEπfV¹) or two (120 μEm^"zs^"1) experiments. Figure 6 is a representation ("Organogenesis Map") of the effects of IBA and kinetin concentrations on organogenesis of TCLs at pH 5.8. Relative mean numbers of each organ type per TCL cultured at various cytokinin and auxin concentration are represented by the radius and proportion of pie. Organ types are represented as follows: roots (\\\), flowers (///), and vegetative shoots (.≡.). Data represent at least two experiments with a minimum of four replicate TCLs per experiment. A dash means' no data were recorded.

Figure 7 is a comparison of TCL explant morphogenesis on media with and without pectic fragments. Figure 7(A) is the scale used for the quantitation of asymmetric (polar) TCL tissue enlargement. Figure 7(B) is a plot of IBA (μM) versus kinetin (μM) comparing the effect of the addition of pectic fragments on TCL morphogenesis. "+ " indicates a difference of greater than 2 organs per TCL. A "P" indicates an average quantitative change in polarity of greater than 0.5 (see A), but with a difference of less than 2 organs per TCL. A"-" indicates no significant effect of adding cell wall fragments. Figure 7(C) is a schematic representation of the types of organs (zones) obtained by culturing TCLs on media containing the indicated concentrations of IBA (μM) and kinetin (μM). R, root zone; F, flower zone; V, vegetative shoot zone; and T, transition zone.

Figure 8 plots number of roots per TCL versus EPGaA4 (μg/ml), demonstrating the inhibition of Root Formation by Pectic Fragments. Mean numbers (± SE) of roots are those formed per TCL after 24-25 days culture on root-inducing medium (15 μM IBA and 0.5 μM kinetin) containing the indicated amounts of EPGaA4 pectic fragments. Data represent four experiments with a minimum of eight replicates per experiment.

Figpre 9 plots number of flowers per TCL versus EPGaA4 (μg/ml), to show induction of flower formation by pectic fragments. Mean numbers (± SE) of flowers are flowers formed per TCL after 24-25 days culture on transition medium (1.5 μM IBA and 0.9 μM kinetin) containing the indicated amount of EPGaA4 pectic fragments. Data represent four experiments with a minimum of five replicates per experiment.

Figure 10 is a plot of number of flowers per TCL versus EPGaA4 or control substance (μg/ml), showing the flower-inducing activity of cell wall fragments is stable to protease digestion and heat treatment. Mean numbers (± SE) of flowers are flowers formed per TCL after 24-25 days culture on transition medium (1.5 μM IBA and 0.9 μM kinetin) containing the indicated amounts of EPGaA4 pectic fragments, heat-treated protease (no wall fragments), or a mixture of monosaccharides (see Table 1 and text). Data represent two experiments with a minimum of six replicates per experiment. Error bars are less than ± 0.67 flowers per TCL. CWF, EPGaA4 fragments; CWFp, protease and heat treated EPGaA4 fragments; CWFh, heat treated EPGaA4 fragments; P, heat treated protease; S, monosaccharide mixture.

Figures 11 A and 11B are plots of the number of organs per TCL for sycamore (A) or (B) tobacco versus pectic cell wall fragments (μg/ml), comparing the abilities of sycamore and tobacco cell wall pectic fragments to induce flowers on tobacco TCLs. Average number of flowers (o) and vegetative shoots ( \) are those formed per TCL after 24-25 days in culture on transition medium (1.5 μM IBA and 0.8 μM kinetin) containing the indicated amount and type of pectic fragments. Data are expressed as mean number of organs formed per TCL (± SE) from two experiments each with six replicates per treatment.

Detailed Description of the Invention

A modified and improved protocol for a plant thin-cell- layer explant culture system which is relatively simple, highly sensitive and, most importantly, reproducible, is disclosed. Specific auxin and cytokinin concentrations have been identified that induce the formation of roots, vegetative shoots, or flowers from TCLs cultured on otherwise identical liquid medium. The TCL culture system has utility as a plant morphogenesis model system that can be used to dissect, into specific developmental and molecular components, the events that occur during plant organ formation. The TCL system is also useful as an assay for the identification, purification, and study of molecules, such as oligosaccharins, that are capable of altering or regulating plant organogenesis and morphogenesis.

The intensity of light illuminating the TCLs and nutrient medium during culture has been discovered to have a dramatic effect on TCL organogenesis. Evidence is provided that the observed effect of light on TGL morphogenesis results from a light-induced degradation of the auxin indol-3-butyric acid (LBA) in the culture medium-. Importantly, variability in TCL morphogenesis due to differences ήt-Hght intensity, which has been a problem in previous TCL systems, can be minimized by culturing the TCLs under a uniform reduced light intensity (e.g., 55 ± 5 mEinstein m^"2sec-l). The improved TCL assay system can be applied to any plant tissue and cell type capable of being cultured as thin-cell-layer explants. Thin-cell-layer explants are routinely cultured on nutrient medium (basal medium) such as that of Linsmaier and Skoog ("Organic Growth Factor Requirements of Tobacco Tissue Cultures", PhysioL Plant 18, 100-127, (1965)) supplemented with 167 mM glucose. However, other nutrient mediums that are capable of maintaining and supporting growth of thin-cell-layer explants in culture can also be used. The nutrient medium is generally adjusted to a pH of approximately 5.8, although any pH can be used that will permit TCL culture growth. Generally, the pH of the medium will fall within the range of 3.5 to 7.0. A particular benefit of this assay is that a single pH can be used to obtain all the types of organogenesis. Further, as will become apparent in view of the preferred embodiments below, variations in plant organogenesis in the TCL system depend only on changes in the concentrations of the phytohormones added to the culture medium. All other factors and variables of the TCL culture system are held constant. The results is that the TCL system can be used to more precisely and reproducibly predict the type and quantity of plant organs formed during culture as a function of concentration of added molecules. This reliability has important benefits as both a model system of plant organogenesis and as an assay tool for identifying and purifying molecules affecting plant morphogenesis.

The assay is useful in screening a variety of molecules affecting organogenesis to determine what effect, if any, the molecule has on organogenesis. For example, pesticides, waste discharges from plants or water treatment plants, or potential toxins can be tested to determine their effect on root, stem or flower growth. The assay can also be used to rapidly screen many compounds for activities which mimic or inhibit organogenic activities. The major classes of phytohormones include cytokinins, auxins, gibberellins, abscisic acid, and ethylene. Auxin and cytokinin are the preferred phytohormones for addition to TCL basal medium although other phytohormones having comparable activities on organogenesis can be substituted for auxin or cytokinin, or added to the medium as a matter of choice. In general, auxin and cytokinin are added to the medium to concentrations of between approximately 0 to 20 μM, depending on the desired plant organogenesis. For example, flower-inducing medium (FM), transition medium 1 (TM1), and transition medium 2 (TM2) consist of basal medium supplemented with 0.5 μM IBA and 0.5 μM kinetin, 4 μM IBA and 0.5 μM kinetin, and 6 μM IBA and 0.5 μM kinetin, respectively.

The following basal medium used for the TCL Morphogenesis Bioassay is as described by Linsmaier and Skoog (1965) except that glucose was used rather sucrose.

Glucose 30.0

The basal medium is made using the following stock solutions: Skoog I 50 ml/L (38.0 g KN0₃; 33.0 g

NH₄NO_s; 7.4 g MgS0₄ ^*7H₂0; 8.8 g CaCl₂2H₂0; 3.4 g KH₂P0₄ dissolved in water at room temperature with stirring; store at 4°C) Skoog^* π 5 ml/L (2.78 g FeS0₄ ^*7H₂0;

3.73 g Na₂EDTA Dissolve the iron sulfate in hot water, approximately 60°C, with stirring; then add EDTA; solution must turn clear gold-yeHow; store at 4°C) Skoog m 1 ml/L (1.24 g H₃B0₃; 4.46 g

MNS0₄'4H₂0; 0.166 g KI; 2.12 g ZnSO 7H_zO; 5.0 ml stock solution of 50 mg/50 ml CuS0₄5H₂0; 0.050 g Na₂Mo0₄2H₂0; 5.0 ml stock solution of 50 mg/50 ml CoCl₂'6H₂0. Dissolve first two salts together in approximately 60°C water, adding other salts individually with stirring as each completely dissolves; store at 4°C)

Thiamine-Inositol 4 ml/L (10.0 mg Thiamine HC1; 2.5 g Myo-Inositol to 100 ml water)

Glucose 30 g/L The TCL cultures are grown under constant light for the assay. Any light source capable of supporting growth of thin-cell-layer explants in culture can be used. Examples of typical light sources include cool white fluorescent light, fluorescent light designed for use in growing plants wherein long wavelengths in the red portion of the visible spectrum are enhanced (Gro³¹ Lights), natural fluorescent light, and incandescent light. Cool white fluorescent light is the preferred light source. The intensity of light provided to the TCL explants, and in particular to the culture medium, is essential. It has been found that high intensity light causes degradation of culture medium components, in particular IB resulting in lower concentrations of biologically active auxin. While light can be supplied to the TCL culture in an intensity range of between 0 and 150 μEπf V¹, cool white fluorescent light with an intensity of 55 μE m^"2s^"1 is preferred. TCL cultures are grown for a period of time ranging from one to 45 days. The resulting culture growth and plant morphogenesis in then measured visually using a dissecting microscope and the specific type and quantity of plant organ or organ primordia is recorded. Plant organogenesis and morphogenesis can also be determined biochemically by assaying the TCL cultures for varying levels of enzymes or enzyme products that are indicative of specific organ development. Similarly, plant organogenesis and morphogenesis can be determined at the molecular level by measuring changes in the level of expression of root-specific, vegetative shoot-specific, or flower- specific genes. For example, RNA-DNA hybridizations in the form of Northern blots, dot blot analysis, or assays using radioactively labeled DNA probes can be performed to quantify the level of expression of particular genes. Such assays are well known to those skilled in the art. Examples of particular enzymes or genes that are useful as indicators of morphogenesis include those related to pigment formation, such as chlorophyll, or to pollen formation. Other genes and enzymes useful as indicators of plant morphogenesis will become apparent to those skilled in the art as a result of further investigation into the biochemical and genetic aspects of plant morphogenesis using the TCL assay system. The TCL assay system contains a number of modifications and improvements over the current state of the art which reduce variability and increase reproducibility. For example, Tran Thanh Van and colleagues (1985) induced different types of TCL organogenesis by altering the pH and the indol butyric acid (LBA) and kinetin concentrations of the culture medium. The present system has been modified and simplified by determining that root, vegetative shoot, and flower organogenesis could be induced in media adjusted to the same pH.

Another important modification of the current state of the art is the recognition that low intensity light (approximately 55 μE m^" V¹) is crucial in order to reduce auxin degradation and increase culture reproducibility.

Additionally, the TCL culture technique of Tran Thanh Van, et al., (1985), entails incubating 20 TCLs in 100 x 15 mm Petri dishes. This practice, however, results in TC s cultured in the same Petri dish occasionally exhibiting greater uniformity in morphogenesis than TCLs in replicate Petri dishes. In contrast, this assay incubates a single TCL per well in 12-well cluster dishes. Incubating TCLs individually provides the additional benefit of reducing TCL loss due to microbial contamination as well as reducing the number of TCLs needed for statistically significant results, since each TCL serves as a replicate rather than a subsample of a single replicate containing 20 TCLs.

The present invention will be further understood by the following non-limiting examples. It will be appreciated that additional uses and modifications will become apparent to those skilled in the art. Such uses and modifications are intended to fall within the scope of the claimed subject matter. EXAMPLE 1: Effect of variation of pH, indoIe-3-butyric acid

(IBA), kinetin, and light on TCL morphogenesis.

METHODS: Plant Material.

Nicotiana tabacum L. cv Samsun line 5 plants (Tran Thanh Van et al., 1985) were grown in a greenhouse at 23-3 l^βC. Supplemental lighting was provided by high pressure sodium lights in order to maintain a 14-hour day length. Plants were grown in a peat- lite soil mix (Fafard No. 3, Conrad Fafard, Springfield, MA) in 20 cm clay pots and fertilized twice each week with Peters 20-20-20 fertilizer (containing 473 ppm N). Since ambient ozone levels were too high for growth of healthy tobacco plants in the greenhouse from late spring to early autumn, air was passed through carbon filters (RSE, New Baltimore, MI) that removed approximately 80% of the naturally occurring ozone from the air. Thin-cell-layer Assay.

The second and third primary floral branches of a typical tobacco inflorescence, illustrated in Figure 1, were cut into 7-8 cm sections when approximately 30% of the flowers had produced green fruits. Floral branches were soaked 2 min in 0.5% Tween 20, surface- sterilized 8 min in 10% commercial bleach (Clorox containing 5.25% NaOQ), and rinsed three times (total of 10 min) in sterile deionized water. Strips of tissue, approximately 1 mm wide and comprised of 1 layer of epidermal cells, 2-3 layers of chlorenchyma and 3-6 layers of collenchyma and/or parenchyma, were cut from the floral branch tissue. TCLs approximately 10 mm long were cut from the tissue strips and floated individually on 2 ml of a Linsmaier and Skoog (1965) medium containing 167 mM glucose (basal medium) and the indicated amounts of indole butyric acid and kinetin (Sigma, St. Louis, MO). The media were filter-sterilized using 0.2 μm filtration units (Nalgene Labware, Rochester, NY). Unless otherwise indicated, the pH of each medium was adjusted to 5.8 by titration with KOH.

TCLs were placed individually in 7 ml wells of 12-well cell culture cluster dishes (Gibco, Grand Island, NY), and the dishes were sealed with two layers of parafilm (American Can Co., Greenwich, CT). The TCLs were incubated at 24°C under cool white (natural or Grolux¹"^* where indicated) fluorescent lights (Sylvania, Danvers, MA) at 55 (±) 5 μEinsteins m^"2sec^"1 (unless otherwise indicated). After 23-25 days of culture, the morphogenesis of TCLs was visually scored using a dissecting microscope. Three types of organs (roots, flowers, and vegetative shoots), the general size, and the degree of polar enlargement of the TCLs were recorded. One organ was defined as the root(s), vegetative shoot(s), or flower(s) that grew from a single initial shoot or root meristem. An individual flower formed directly on the TCL surface was recorded as one flower, as was an inflorescence having five flowers. Likewise, a primary root with three lateral roots would be recorded as one root. The data were analyzed using an R:Base for DOS database (Microrim, Redmond, WA). Quantitation of IBA in the Medium.

The basal medium containing 6 μM IBA and 0.5 μM kinetin was incubated in cluster dishes under cool white fluorescent light at 55 or 120 μEm^'V¹. Dishes covered with aluminum foil served as dark controls. IBA concentration was determined by gas chromatographic (GC) analysis of duplicate samples collected from each treatment after 0, 2, 4, 6, 12, and 24 days of incubation. IBA was extracted from the medium by partitioning with ethyl acetate (HPLC grade). An internal standard of 1 μg indole-3-propionic acid (IPA) was added to each sample after the extraction. Indoles were converted to trimethylsilyl (TMS) derivatives by heating them at 65°C for 20 min with N-0 bis(trimethylsilyl) trifluoroacetamide (BSTFA). The TMS derivatives were analyzed on a Hewlett Packard 5880A series gas chromatograph using a DB-1 30-meter (0.25 mm i.d.) fused silica capillary column (0.25 μm film thickness). The GC oven was programmed from 90°C to 240°C at 15°C min^"1. Injections were made in the splitless mode at 250°C. Quantitative determinations were derived from peak areas using a standard curve for IBA. RESULTS: Effect of varying pH on TCL. Culture of TCLs in 55 μEinsteins m^"2sec^"1 (uEm^'V¹) light on a medium containing 0.5 μM IBA and 0.5 μM kinetin resulted in a mixture of flowers and vegetative shoots, regardless of the pH of the medium, as shown in Figure 2A. Very few organs were formed when TCLs were incubated on media containing 5.0 μM IBA, except at pH 6.15 where flowers were produced (Figure IB). Roots were formed at each pH tested on a medium containing μM IBA and 0.5 μM kinetin (Figure 2C). A slightly greater number of flowers and roots were formed on TCLs cultured on media that had been adjusted to a pH between 5 and 5.8. Similar pH effects were obtained in duplicate experiments with TCLs incubated in 95 μEm^"2s^"1 light.

These results demonstrated that, with the exception of the formation of flowers at pH 6.15 on medium containing 5 μM IBA variation of pH within the range originally used by Tran Thanh Van et al., (1985) caused only small changes in the number of organs formed on TCLs and had no deciding influence on the type of organ formed. These results demonstrated that altering the pH of the medium is not required to obtain the three types of TCL organogenesis. As a results of this study, the TCL assay in cluster dishes was further modified and simplified in subsequent studies by adjusting each medium to pH 5.8, a pH within the range commonly used for plant tissue culture. Effect of light Quality on TCL Organogenesis.

In initial experiments TCLs were cultured under cool white fluorescent Jljghts supplemented at intervals with incandescent lights as in Cousson aιt_d Tran (1983). Due to concerns that the variable positioning of incandescent lights would produce unequal light quality for the TCLs, the use of the fluorescent light alone was tested to determine if it could be used to obtain root, vegetative shoot, and flower organogenesis.

TCLs were cultured on basal medium containing various concentrations of IBA and kinetin and incubated at 50 μEm^"2s^"1 under one of three types of Sylvania fluorescent lights: cool white, Grolux™, or natural. AQ, types of organogenesis, as shown in Table 1, were induced using fluorescent light without incandescent light as a supplement Based on these results, the TCL culture protocol was further modified such that subsequent experiments were performed using only cool white fluorescent light

Cool white (CW), Grolux™ (GL), or natural (Nat) fluorescent lights. Twelve TCLs were grown under each light condition.

Mean numbers of flowers (F), vegetative shoots (VS), or roots (R) from N TCLs ± SE.

Change in Light Intensity Alters TCL Organogenesis.

Growth of 20 TCLs per Petri dish under the different light intensities shown in Figure 3 was confirmed to dramatically alter organogenesis of TCLs cultured in Petri dishes. When TCLs were incubated on a medium containing 0.5 μM IBA and 0.5 μM kinetin at pH 3.8, the number of flowers decreased as the light intensity was increased (Figure 3A). TCLs incubated on a medium containing 3 μM IBA and 05 μM kinetin at pH 6.15 (Figure 3B) formed roots at the lowest light intensity (55 μEm^'Y ), vegetative shoots at the middle light intensities (75 and 95 μEm^'V¹), and flowers at the two highest light intensities (95 and 115 μEm^"2s^"1). The number of roots formed by TCLs cultured on a medium containing 7 μM IBA and 0.2 μM kinetin at pH 5.8 decreased at the higher light intensities (Figure 3C). In vitro assays, such as the TCL assay, are most useful for studies of organogenesis and other aspects of morphogenesis if factors that cause irreproducibility have been identified and controlled. Accordingly, tests were conducted to determine how sensitive TCL organogenesis was to light quantity in the modified system of incubating TCLs at pH 5.8 in cluster dishes, using cool white fluorescent light

TCLs were incubated under 55, 75, 95 and 120 (± 5) μEm^"2s^"1 light on a flower-inducing medium, a transition medium (TCLs cultured on transition medium form few or no organs), a root- inducing medium, and a vegetative shoot-inducing medium. Increasing the light intensity caused a decrease in the number of flowers when TCLs were cultured on the flower-inducing medium, as shown in Figure 4A. This effect was similar to that observed in the Petri plate experiment (compare Figure 4A to Figure 3A). More flowers were produced under the highest light intensity than under the lowest light intensity when TCLs were incubated on transition medium (Figure 4B). Conversely, fewer roots formed on TCLs incubated on root-inducing medium under the highest light intensity than under the lowest light intensity (Figure 4C). Light intensity did not have a significant effect on vegetative shoot formation when TCLs were incubated on vegetative shoot-inducing medium (Figure 4D).

The Effect of Light Intensity on TCL Organogenesis: Correlation with the Light-Induced Degradation of a Medium Component.

The sensitivity of TCL organogenesis to light could have resulted from a direct physiological response of TCLs to light or, alternatively, as a secondary response to light-induced alteration or degradation of a critical medium component. The latter possibility was tested by comparing the organogenesis of TCLs cultured on fresh media to the organogenesis of TCLs cultured on media that had been preincubated either in the dark or in light. A flower-inducing medium (FM) and two transition media (TMl and TM2) were preincubated for six days at 55 and 120 μEm^"2s^"1 in unwrapped (light-pretreated) and aluminum foil-wrapped (dark-pretreated) cluster dishes. After preincubation of the media, TCLs were placed on pretreated or fresh media and incubated under 55 or 120 μEm^'V¹ light. The types of organogenesis differed when TCLs were cultured on fresh versus light-pretreated media, as shown in Tables 2 and 3. For example, TCLs incubated under 55 μEm^"2s^"1 (Table 2) or 120 μEm^'V¹ (Table 3) light on fresh FM medium or on dark- pretreated FM medium produced flowers. However, no flowers formed on TCLs cultured on the light-pretreated FM medium. The decreased number of flowers produced on TCLs incubated on the light-pretreated FM medium was similar to the reduced number of flowers produced when TCLs were incubated under high light intensities on a flower-inducing medium as indicated above (Figure 4A).

A similar correlation was observed between the changes in organogenesis that occurred when TCLs were cultured on light- -20-

pretreated TMl and TM2 media (Tables 2 and 3) and the changes in organogenesis that occurred when TCLs were cultured on transition medium at increasing light intensities (Figures 3 and 4). For example, TCLs formed roots when incubated at 55 μEm^" s^"1 on fresh or dark- pretreated TM2 medium (Table 2). However, TCLs cultured on the light-pretreated TM2 medium formed flowers rather than roots (Table 2). This change in organogenesis is similar to the change from formation of roots to formation of flowers that occurred on TCLs incubated on transition medium at increasingly higher light intensities as discussed above (Figure 3B).

These results demonstrate that the changes in TCL organogenesis that occurred when TCLs were incubated at increasingly higher light intensities (see Figures 3 and 4) could be mimicked by preincubation of the media in light. These results strongly suggested that the light-induced changes in TCL organogenesis were due to alteration or degradation of a medium components).

Table 2. Effect of Preincubation of Media in ight on

Organogenesis of TCLs Cultured at 55 μEm^"2s^"

Basal medium supplemented with 0.5 μM IBA and 0.5 μM kinetin (FM), 4 μM IBA and 0.5 μM kinetin (TMl), or 6 μM IBA and 0.5 μM kinetin (TM2).

TCLs were cultured for 19 days (expt 1) or 24 days

(EXPT 2).

Twenty TCLs were each incubated in fresh medium

(Control), medium pretreated for 6 days in foil-wrapped cluster dishes placed under 55 μEm^"2s^"1 light (Dark), medium pretreated for 6 days in 55 μEm^'Y¹ light (55), or medium pretreated for 6 days in 120 μEπTY¹ light

(120).

Mean numbers ± SE of flowers (F), vegetative shoots

(VS\ and roots (R). Table 3. Effect of Preincubation of Media in Light on

Organogenesis of TCLs Cultured at 120 μEm^"2sec^"1.

Expt" Treat- Media^a # ment^d F ± SE^d VS ± SE R ± SE

FM

FM

Basal medium supplemented with 0.5 μM IBA and 0.5 μM kinetin (FM), 4 μM IBA and 0.5 μM kinetin (TMl), and 6 μM IBA and 0.5 μM kinetin (TM2). TCLs were cultured for 19 days (expt 1) or 24 days (expt

2)*

Fresh medium (Control), medium pretreated for 6 days in foil-wrapped cluster dishes placed under 55 μEm^~2sec^~1 light

(Dark), medium pretreated for 6 days in 55 μEm^"2sec^"1 light (55), medium pretreated for 6 days in 120 μEm^"2sec^"1 light (120).

Mean numbers ± SE of flowers (F), vegetative shoots (VS), and roots (R). Comparison of the organogenesis of TCLs incubated under 55 versus 120 μEπf^2"1 light (Figure 4) with the types of organogenesis obtained by culturing TCLs on media with a variety of IBA and kinetin concentrations at 55 μEm^"2s^"1 (see Figure 6) showed that a decrease in the amount of IBA in each medium tested could cause the observed light-induced changes in organogenesis. It has been shown that indole acetic acid (IAA) is unstable in light (Yamakawa et al., 1979). It was therefore determined whether incubation of medium in 55 and 120 μEπfY¹ of cool white fluorescent light caused degradation of IBA in the medium.

Effect of Light Irradiation on Stability of IBA in the Medium.

The stability of IBA in the medium was determined by measuring the amount of IBA remaining in basal medium containing 6 μM IBA and 0.5 μM kinetin after the medium was placed in cluster dishes and maintained for various lengths of time under low (55 μEm^{" 2}s^"1) or high (120 μEm^"2s^"1) light intensity. Medium in cluster dishes wrapped in aluminum foil served as light-free (dark) controls. The time-dependent decrease in the concentration of IBA in medium exposed to 55 and 120 μEm^"V¹ cool white fluorescent light is shown in Figure 5. The IBA concentration in medium was reduced by 50% and 88% after 6 days of incubation under 55 and 120 μEm^"2s^"1 light, respectively. The concentration of IBA was reduced by greater than 98% in the medium incubated at either light intensity for 24 days. However, the IBA concentration of the medium in cluster dishes covered with aluminum foil decreased by only 9-12% after 24 days of incubation.

The decreased amount of IBA remaining in the medium incubated under 55 and 120 μEm^"2s^"1 light correlates with the changes in organogenesis that occurred when TCLs were cultured on light- pretreated media (Tables 2 and 3). Thus, light-induced degradation of IBA appears to be largely or entirely responsible for the observed light-induced changes in TCL organogenesis. TCLs were incubated under 55 (± 5) μEm^'V¹ cool white fluorescent light for subsequent studies in order to additionally simplify the TCL assay and reduce the number of variable experimental parameters.

Organogenesis in Thin Cell Layers can be Determined by the Concentrations of IBA and Kinetin in the Medium.

TCLs were cultured on media containing a range of IBA and kinetin concentrations in order to characterize the quality, quantity, and reproducibility of TCL morphogenesis using our modified and simplified culture conditions. A TCL "organogenesis map" of organ formation in response to various IBA and kinetin concentrations is illustrated in Figure 6. When pH and light intensity were held constant, the IBA and kinetin concentrations in the media were sufficient to determine whether TCLs formed roots, vegetative shoots, or flowers. Roots formed when TCLs were cultured on media with relatively high IBA (5-20 μM) and low kinetin (0.1-0.75 μM) concentrations. Vegetative shoots formed when TCLs were cultured on media with high kinetin (2-10 μM) and a range of IBA concentrations. Flowers formed when TCLs were cultured on media with low IBA (0.1-2 μM) and low kinetin (02-2 μM) concentrations. Media with IBA and kinetin concentrations falling between those concentrations that resulted in the formation on TCLs of roots, vegetative shoots, or flowers are referred to as transition media.

TCLs cultured on transition media formed few or no vegetative shoots, flowers, or roots. IBA and Kinetin can Induce Asymmetric Organogenesis and Tissue Enlargement of TCLs.

Hormone-dependent differences in the polarity of organogenesis and tissue enlargement of TCLs incubated on media with a range of IBA and kinetin concentrations can be compared by visual examination. Polar enlargement and polar organ formation, if it occurs, is always at the basal end of the TCL (the end that had been nearest the stem). The orientation of the TCLs after 24 days of culture was recognized by marking the basal or apical end of the TCL with a diagonal cut at the time the explant was removed from the floral branch, or, alternatively, by maintaining the TCL's orientation during the course of the experiment by growing the TCLs on paper wicks kept in contact with the medium.

Based on observations of TCLs cultured on media with a range of IBA and kinetin concentrations, the following general conclusions can be made:

(1) Pronounced polar enlargement occurs on TCLs cultured on media containing less than 5 μM kinetin, while uniform enlargement occurs when the concentration of either IBA or kinetin in the media exceeds 5 μM; and

(2) organs form either predominantly on the basal end of the TCL or over the entire TCL, depending upon the organ type and the IBA and kinetin concentrations in the medium.

(3) TCLs cultured on root-inducing medium usually form roots with a nonpolar ("random") distribution, except on root-inducing medium with relatively low levels (e.g., 4 μM) of IBA (see Figure 6). Vegetative shoots form in a nonpolar fashion, except on vegetative shoot-inducing media containing less than 3 μM kinetin. Flowers form in a polar manner, but this polarity is reduced on media with greater than 1 μM kinetin. (4) The polar distribution of organ formation is not always accompanied by polar tissue enlargement. For example, roots can form in a polar manner on TCLs that show little or no polar tissue enlargement (4 μM IBA and 0.1 μM kinetin), and flowers can form in a nonpolar fashion on TCLs that have well-defined polar tissue enlargement (1 μM kinetin). Thus, IBA and kinetin are able to regulate the polarity of tissue enlargement and organogenesis. This improved TCL assay system enables one skilled in the art to observe and predict the effect that changes in phytohormone concentrations, particularly auxin and cytokinin concentrations, as well as concentrations of other molecules, will have on TCL organogenesis. The improved TCL system has been used to develop a IBA-kinetin "organogenesis map" to characterize the effect of various parameters on TCL organogenesis, shown in Figure 6.

TCLs cultured on transition media are particularly useful in deciphering the effects of light on organogenesis, since relatively small changes in the concentration of a hormone resulted in an easily observed change in organogenesis. Similarly, the sensitivity of TCL's cultured on transition medium is particularly useful in screening exogenously added compounds for the ability to affect plant morphogenesis. Example 2: Use of the improved TCL assay system to confirm that plant oligosaccharins can regulate TCL morphogenesis.

Oligosacehώin-containing pectic fragments of plant cell wall polysaccharides, released from the walls of suspension-cultured sycamore cells ^"(Acer pseudoplatanus) by treatment with endopolygalacturonase, were tested for morphogenesis-regulating activity in a tobacco thin-cell-layer explant (TCL) assay system. The pectic fragments inhibited the formation of roots on TCLs grown on root-inducing medium containing 15 μM indole-3-butyric acid (IBA) and 05 μM kinetin. Addition of the pectic fragments to a root- inducing medium containing 7 μM IBA and 0.15 μM kinetin caused roots to form on the extreme basal end of TCLs. TCLs cultured on this medium in the absence of added pectic fragments form roots with no preferred location. The pectic fragments induced polar tissue enlargement and the formation of flowers in TCLs cultured on transition medium. The flower-inducing activity was stable to heat treatment and protease digestion indicating that the active agent was not proteinaceous in nature. Pectic fragments isolated from the walls of suspension-cultured tobacco cells (Nicotiana tabacum L. cv Samsun) were as effective as pectic fragments from the walls of sycamore cells in inducing flowers to form on the TCLs. These results indicate that oligosaccharins from plant cell walls are able to regulate morphogenesis.

The plant cell wall is a dynamic structure that serves both structural and functional roles in plants. It has been shown that pectic polysaccharide fragments from the plant cell wall act as endogenous elicitors which induce plants to protect themselves against pathogens. Fragments of plant cell wall polysaccharides have also been shown to regulate growth and development of plants. Fragments produced by partial acid hydrolysis of cell walls have been shown to inhibit flowering and promote vegetative growth in Lemna gibba. These results suggest that the cell wall might be a repository of chemical signals that regulate plant development.

Support for the hypothesis that cell wall fragments function in the control of plant development was obtained using the in vitro thin-cell-layer explant (TCL) assay system of Example 1. The effects of endopolygalacturonase-related cell wall fragments on plant cell morphogenesis in the TCL assay system are presented. METHODS: Plant Material.

Tobacco plants (Nicotiana tabacum L. cv Samsun) were grown as described in Example 1. Thin-Cell-Layer Assay.

The TCL assay procedure was used as described above for Example 1. When pectic fragments were tested, a stock solution was filter-sterilized using an 0.2 μm nylon membrane syringe filter (Nalgene) and aseptically added to the medium. Plant Cell Wall Isolation.

Cultures of sycamore cells (Acer pseudoplatanus) originally isolated by D.T.A. Lamport in 1958 have been maintained in the dark since 1960. The cells are grown on the modified M-6 medium of Torrey and Sbigemura, Am. J. Bot. 44, 334-344 (1957) and subcultured to fresh medium every 7 days. Primary cell walls of suspension- cultured sycamore cells were isolated as described by Talmadge et al., Plant Phvsiol. 51, 158-173 (1973).

A tobacco suspension culture was established from freshly isolated pith callus of the same variety of tobacco used in the TCL system (Nicotiana tabacum L. cv Samsun). The cultured cells were grown in the dark on an LS medium, Linsmaier and Skoog, Phvsiol. Plant. 18, 100-127 (1965) supplemented with 1 mg 1 2,4- dichlαrophenoxyacetic acid and 30 g sucrose per liter. The cells were subcultured to fresh medium every 14 days. Purification of Endopolygalacturonase.

Eήdo-α-l,4-polygalacturonase was purified to homogeneity from a commercial preparation of Aspergillus niger pectinase by carboxymethylcellulose chromatography, preparative isoelectric focusing, and gel-permeation chromatography on Sephadex G-50, as described by Cervone et al., Plant Phvsiol. 85, 626-630 (1987). Comparative studies were done using an endo-a-1,4- polygalacturojiase from Colletotrichum lindemuthianum prepared as described (English et al, Plant Phvsiol. 49, 293-297 (1972); York et al., Methods in Enzymology 118, 3-40 (1985)), and an endo-a-1,4-

RMzopus arrhizus purified from a commercial preparation of pectinase (Sigma # P2401) by a modification of the method of Lee and West, Plant Phvsiol. 67, 633-639 (1981). Isolation of Pectic Cell Wall Fragments.

Pectic polysaccharides were extracted from purified walls of

sycamore and tobacco cells by digestion of the cell walls with endo-a-l,4-polygalacturonase purified from A. niger by the procedure described by York et al., (1985). Glycosyl-Composition Analysis of Pectic Fragments.

Glycosyluronic acid and neutral glycosyl residues of EPGaA4 pectic fragments were simultaneously quantitated as their per-Otrimethylsilyl methyl glycosides, prepared and analyzed as described by Chambers and Clamp, Biochem. J. 125, 1009-1018 (1971); York et al., (1985)). Protease Treatment of Pectic Fragments.

EPGaA4 pectic fragments (1 mg in 1 ml H₂0) were incubated with 1 unit Pronase (Boehringer Mannheim) for 1 hr at 37°C and heated at 120°C for 10 min to destroy enzyme activity. The protease activity of the Pronase-cell wall fragment solution, both before and after the 120°C treatment, was measured as Azocoll- digesting activity using the method of Ragster and Chrispeels, Plant Phvsiol. 64, 857-862 (1979). A 50 μl aliquot of the Pronase-treated cell wall fragment solution was incubated with 15 mg of Azocoll

(Calbiochem) in a total volume of 1.5 ml for 1 hr at 37°C followed by incubation at 4°C for 15 min. The reaction mixture was then centrifuged for 5 min at 15,600 x g and the absorbance at 520 nm recorded. RESULTS:

Pectic Fragments of Cell Walls Alter TCL Morphogenesis.

A large scale preparation of endopolygalacturonase- released wall fragments from suspension-cultured sycamore cells was used to screen media containing a variety of IBA and kinetin concentrations for the ability of the pectic fragments to alter TCL morphogenesis. The glycosyl composition of this pectic fragment preparation (EPGaA4), shown in Table 4, is similar to that of other endopolygalacturonase-related pectic fragment preparations. Amino acids, if present, were below the limits of detection of heptafluorobutyric anhydride amino acid derivatives as analyzed by gas chromatography. All results were obtained using EPGaA4 wall fragments; however, other identically prepared sycamore cell wall pectic fragments had the same ability to alter TCL morphogenesis.

The effect of pectic fragments on TCL morphogenesis was first screened by culturing TCLs on media containing a range of LBA and kinetin concentrations in the absence and presence of 10 μg/ml EPGaA4. The number and type of organs on 24/25-day-old TCLs, as well as the symmetry of tissue enlargement, were recorded by examining each TCL in every experiment with a dissecting microscope. Representative TCLs were photographed for a permanent visual record. The scale for assessing asymmetric (polar) enlargement of the TCLs is illustrated in Figure 7A A value of 0 represents symmetric enlargement, 1 represents asymmetric enlargement, which results in a TCL of more or less triangular shape, and 2 represents polar enlargement As discussed above polar enlargement occurs at the basal end of the TCL -

The addition of pectic fragments affected TCL morphogenesis differently depending upon the concentration of IBA and kinetin in the medium (Figure 7B). Pectic fragments caused a change in the number of organs formed (+), increased polar tissue enlargement (P), or had no effect on morphogenesis (-). Comparison of Figures 7B and 7C reveals that the most pronounced effect of endopolygalacturonase-released pectic fragments occurred on root- inducing and transition media. On transition media, TCLs form either no organs or very few vegetative shoots, flowers, or roots. These fragments had no identifiable effect on TCLs cultured on flower- inducing and vegetative shoot-inducing media. Several changes were observed on TCLs cultured on root-inducing and transition media. Addition of pectiαjfragments to root-inducing and transition media generally caused an increase in asymmetric (polar) enlargement of the TCLs_* In some media, the change in polarity was accompanied by a change in the number, type, or distribution of organs. Four media were used for further analysis of the pectic fragment effects. The hormone concentrations of these media and the effects on TCL morphogenesis of the addition of pectic fragments at a concentration of 10 μg/ml are summarized in Table 5 and discussed in more detail below. Pectic Cell Wall Fragments Alter Morphogenesis of TCLs Cultured on Root-Inducing Medium.

Addition of pectic fragments to a root-inducing medium containing 15 μM IBA and 0.5 μM kinetin resulted in inhibition of root formation on TCLs. Increasing the concentration of pectic fragments from 0.1 to 10 μg/ml resulted in increasing inhibition of root formation, as shown in Figure 8. Inhibition of root formation was accompanied by a decrease in the size of the TCL and more pronounced polar tissue enlargement.

Pectic fragments added to a root-inducing medium that contained 7 μM IBA and 0.15 μM kinetin changed the location, but not the number of roots formed on the TCLs. Addition of pectic fragments at a concentration of 10 μg/ml to TCLs cultured on a medium containing 7 μM IBA and 0.15 μM kinetin caused approximately 90% of the roots to form at the basal end of the TCLs. Only approximately 25% of the roots formed in this asymmetric manner on TCLs cultured on the same medium without the addition of pectic fragments; that is, the location of roots was essentially random. The polar formation of roots in the presence of pectic fragments was accompanied by polar enlargement of the TCLs. Pectic Cell Wall Fragments Alter Morphogenesis of TCLs Cultured on Transition Medium.

TCLs incubated on a transition medium containing 4 μM IBA and 0.5 μM kinetin formed no organs or, on average, less than one flower, root, or vegetative shoot per TCL. Addition of pectic fragments at a concentration of 10 μg/ml to the transition medium caused asymmetric (polar) tissue enlargement at the basal end of the TCLs. The addition of pectic fragments at a concentration of 10 μg.ml completely inhibited any roots that would have otherwise foπned on this medium. Sometimes a concomitant induction of a few flowers or vegetative shoots was observed.

The formation of numerous flowers was observed when pectic fragments were added to a transition medium that contained 1.5 μM IBA and 0.9 μM kinetin. Addition of pectic fragments at a concentration of 0.1- 10 μg/ml resulted in a concentration-dependent increase in the- number of flowers formed per TCL, as shown in

Figure 9.

Initial Characterization of the Pectic Cell Wall Fragments That Exhibit Morphogenesis-Altering Activity.

The results described above demonstrate that addition of pectic fragments to the culture medium causes four different types of reproducible changes in TCL morphogenesis, and that each particular change is a function of the concentrations of auxin and cytokinin in the medium. The EPGaA4 pectic fragments were prepared by treatment of sycamore cell walls with an apparently homogeneous endopolygalattturonase purified from the culture medium of the fungus Aspergillus ni^ef. ^* The possibility that the morphogenesis-altering activity was a component inadvertently introduced with the Aspergillus enzyme was tested by treatment of sycamore cell walls with endopolygalacturonases isolated and purified from two other fungi, Rhizopus arrhuus and Colletotrichum Undemuthianum. Three pectic fragment preparations of sycamore cell walls were prepared by treating separate wall samples with the three fungal endopolygalacturonases. Each of the three fragment preparations was able to inhibit root formation of TCLs cultured on root-inducing medium and to inhibit root formation, induce polar growth, and occasionally induce a few flowers or vegetative shoots on a transition medium (4 μM IBA and 0.5 μM kinetin). These results are consistent with the hypothesis that pectic oligo- or polysaccharides in EPGaA4 are responsible for its morphogenesis-altering activity.

To rule out the possibility that very small amounts of protein may have been present in EPGaA4, even though no amino acids were detected in this preparation, it was determined whether the morphogenesis-altering activity resided in a protein or heat-sensitive enzyme by incubating EPGaA4 fragments for 1 hr at 37°C with sufficient amounts of a general protease (Pronase) to degrade a protein sample of a mass equivalent to the total mass of the pectic fragment sample. The fragments were then heated at 120°C for 10 min to inactivate the protease. Other EPGaA4 fragments were subjected only to the heat treatment. The flower-inducing activity of the pectic fragments was stable to both the heat treatment and the protease digestion, as illustrated in Figure 10. Heat-inactivated protease by itself did not induce flower formation, nor did a mixture of monosaccharides equivalent to the molar glycosyl composition of EPGaA4 (see Table 4, Figure 10). These results indicate that the active component in EPGaA4 was a pectic oligo- or polysaccharide of cell wall origin.

Light degrades IBA in the TCL assay medium, as discussed above. Measurement of the amount of IBA remaining in the medium, incubated for 12 days under 55 μEinsteins m^"2sec^"1 light, with and without pectic fragments at a concentration of 10 μg/ml, showed that pectic fragments had no effect on IBA stability in the medium. Both with and without fragments, 80% of the IBA added to the medium was degraded. Also, equilibrium dialysis indicated that no detectable binding occurred between the pectic fragments and IBA in the medium. Pectic Fragments of Tobacco Cell Walls Induce Flowers to Form on the TCLs.

The results described above establish that pectic fragments isolated from the walls of suspension-cultured sycamore cells are able to alter morphogenesis of tobacco explants. If cell wall fragments serve as regulators of morphogenesis in vivo, then fragments of tobacco cell walls should also be able to regulate morphogenesis in tobacco explants. To examine this question, pectic fragments were isolated from the walls of suspension-cultured tobacco cells derived from the same variety of tobacco as that used in the TCL assay. The ability of pectic fragments from tobacco cell walls to induce flowers on TCLs, cultured on 1.5 μM IBA and 0.8 μM kinetin, is shown in Figure 11B and is comparable to the ability of pectic fragments from sycamore cell walls Jo induce flowers, which is shown in Figure 11 The minor difference between the number of flowers induced by sycamore and tobacco pectic fragments is no greater than that observed using different preparations of sycamore wall pectic fragments. These results demonstrate that the TCL assay system can be used to screen for compounds capable of affecting morphogenesis of plant tissues and cells. Specifically, the improved TCL assay system was successfully employed to provide evidence that pectic cell wall fragments isolated from sycamore and tobacco suspension-cultured cells can regulate tobacco TCL morphogenesis.

Table , Glycosyl Composition of EPGaA4 Fragment Mixture^*

Glycosyl Normalized residue mol %

Glycosyl residues < 1% of total are not included in this data. Table 5. Effect of Pectic Cell Wall Fragments (10 μg/ml) on Organogenesis and Polarity of TCLs

Effect IBA μM Kinetin μM

Inhibition of root formation Induction of polar root formation Induction of polar tissue enlargement Induction of flower formation

Claims

We claim:

1. A method of assaying for molecules affecting plant morphogenesis comprising:

(a) culturing thin-cell-layer explants of plant origin in a nutrient medium having a uniform pH in the range of approximately 35 to 7.0;

(b) providing the thin-cell-layer explants with a flux of light having an intensity of between approximately 0 and 150 μEinsteins m^"2 sec^"1;

(c) adding to the nutrient medium an auxin to a concentration range of between approximately 0 μM to 50 μM;

(d) adding to the nutrient medium a cytokinin to a concentration range of between approximately 0 μM to 50 μM;

(e) adding to the nutrient medium molecules to be assayed for effect on morphogenesis such that the agent contacts the thin-cell- layer explants in culture;

(f) culturing the thin-cell-layer explants in contact with the molecules; and

(g) determining if morphogenic changes, including changes in floral meristems, vegetative meristems, root meristems, and size and shape of tissus, have occurred in the cultured plant cells.

2. The method of claim 1 wherein the nutrient medium is selected from the group consisting of medium containing between approximately 8 and 15 μM IBA and between approximately 0.3 and 0.5 μM kinetin; medium containing between approximately 7 and 8 μM IBA and approximately 0.15 μM kinetin; medium containing between approximately 4 and 6 μM IBA and approximately 0.5 μM kinetin; and medium containing approximately 1.5 and 2 μM IBA and between approximately 0.75 and 0.90 μM kinetin.

3. The method of claim 1 wherein the nutrient medium has a pH of approximately 5.8.

4. The method of claim 1 wherein the light is provided at an intensity of approximately 55 μEinsteins m^"2sec^"1'

5. The method of claim 1 wherein the light is selected from the group consisting of cool white light fluorescent light, fluorescent light having a spectrum of light emission enhanced in the red wave lengths for improved plant growth, natural fluorescent light, and incandescent light.

6. The method of claim 1 wherein the auxin is provided in a concentration between 0 and 20 μM and is selected from the group consisting of indole-3-butyric acid, indole-3-acetate, indole-3-propionic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), naturally derived compounds having auxin-like activity in thin-cell-layer cultures, and synthetic analogs of auxins having auxin-like activity in thin-cell-layer cultures.

7. The method of claim 1 wherein the cytokinin is provided in a concentration between 0 and 20 μM and is selected from the group consisting of kinetin, zeatin, lupinic acid, naturally derived compounds having cytokinin-like activity in thin-cell-layer cultures, and synthetic analogs of cytokinin having cytokinin-like activity in thin-cell-layer cultures.

8. The method of claim 1 wherein the molecule is selected from the group consisting of natural and synthetic gibberellins and gibberellin-like compounds, natural and synthetic abscisic acids and abscisic acid-like compounds, pesticides, and toxins.

9. The method of claim 1 further comprising measuring the number, type and distribution of formed plant organs and structures selected from the group consisting of flowers, organs, roots organs, basal enlargement structures, and vegetative shoots organs.

10. The method of claim 1 wherein the thin-cell-layer explants to be cultured are derived from Nicotiana tabacum.

11. The method of claim 1 wherein the plant cell cultures are incubated for between one and 45 days.

12. The method of claim 1 wherein the assay for agents affecting plant morphogenesis comprises:

(a) culturing Nicotiana tabacum plant cells as thin cell layers in transition medium having a uniform pH in the range of approximately 5.8;

(b) providing the plant cells with a constant flux of cool white fluorescent light having an intensity of 55 μEinsteins m^"2sec^"1;

(c) adding to the nutrient medium indole-3-butyric acid to a concentration range of between approximately 0.1 μM to 20 μM;

(d) adding to the nutrient medium kinetin to a concentration range of between approximately 0.1 μM to 10 μM;

(e) adding to the nutrient medium a potential morphpgenesϊt agent such that the agent contacts the plants cells in culture;

(f) culturing the plant cells in contact with the potential morphogenesis agent for a period of approximately 25 days; and

(g) determining if morphogenic changes have occurred in the cultured plant cells.

13. A composition for assaying for molecules affecting plant morphogenesis comprising:

(a) apparatus for growing multiple thin cell-layer explants containing nutrient medium having a uniform pH in the range of approximately 35 to 7.0, wherein said medium is divided into groups containing an auxin in a concentration range of between 0 μM to 50 μM; and

(b) a light source supplying an intensity of light of between approximately 0 and 150 μEinsteins m^"2 sec^"1.

14. The composition of claim 17 further comprising plant thin-cell-layer explants capable of being cultured in the apparatus.

15. The composition of claim 18 wherein the plant cells are derived from Nicotiana tabacum.

16. The composition of claim 17 wherein the nutrient medium is selected from the group consisting of medium containing between approximately 8 and 15 μM IBA and between approximately 0.3 and 0.5 μM kinetin; medium containing between approximately 7 and 8 μM DBA and approximately 0.15 μM kinetin; medium containing between approximately 4 and 6 μM IBA and approximately 0.5 μM kinetin; and medium containing approximately 1.5 and 2 μM IBA and between approximately 0.75 and 0.90 μM kinetin.

17. The composition of claim 17 wherein the nutrient medium has a pH of approximately 5.8.

18. The composition of claim 17 wherein the light source is provided at an intensity of approximately 55 μEinsteins m^"2sec^"1.

19. The composition of claim 17 wherein the light is selected from the group consisting of cool white fluorescent light, natural fluorescent light having a spectrum of light emission enhanced in the red wave lengths for improved plant growth, and incandescent light

20. The composition of claim 17 wherein the auxin is provided in a concentration of between 0 and 20 μM and is selected from the group consisting of indole-3-butyric acid, indole-3-acetate, indole-3-propionic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), naturally derived compounds having auxin-like activity in thin-cell- layer cultures, and synthetic analogs of auxins having auxin-like activity in thin-cell-layer cultures.

21. The composition of claim 17 wherein the cytokinin is provided in a concentration of between 0 and 20 μM and is selected from the group consisting of kinetin, zeatin, lupinic acid, naturally derived compounds having cytokinin-like activity in thin-cell-layer cultures, and synthetic analogs of cytokinin having cytokinin-like activity in thin-cell-layer cultures.

22. The composition of claim 17 wherein the molecule is selected from the group consisting of natural and synthetic gibberellins and gibberellin-like compounds, natural and synthetic abscisic acids and abscisic acid*4ike compounds, pesticides, and toxins.

23. ^'The composition of claim 17 comprising:

(a) Nicotiana tabacum cells capable of being cultured in thin cell layers;

(b) transition medium having a uniform pH of approximately 5.8 containing indole-3-butyric acid at a concentration of between approximately 0.1 μM to 20 μM and kinetin at a concentration range of between approximately 0.1 μM to 10 μM; and;

(c) a cool white fluorescent light source providing a constant flux of light having an intensity of approximately 55 μEinsteins m^"zsec^"1.