Dominick V. Basile

Vitamin B12-stimulated growth and development of Jungermannia leiantha Grolle and Gymnocolea inflata (Huds.) Dum. (Hepaticae)

Exogenous vitamin B,2 supplied to axenically cultured gametophytes of Jungerman- nia leiantha Grolle (Jungermanniaceae) and Gymnocolea inflata (Huds.) Dum. (Lophoziaceae) significantly stimulated growth in both species. Pink-pigmented, facultative methylotrophic bacteria, which synthesize and accumulate vitamin B,2, have been regularlyfound associated with bryophytes. Our findings and those of others cited herein indicate that if the vitamin B2, synthesized by these bacteria, or from any other source, should become available, bryophytes and other embryophytes could benefit.

The Occurrence of Cell Wall-Associated Arabinogalactan Proteins in the Hepaticae

Arabinogalactan proteins (AGPs) constitute a class of hydroxyproline-containing gly- coproteins. Accumulated evidence that hydroxyproline-containing protein(s) (hyp-protein) functions in the correlative control of leaf and branch development in liverworts prompted our investigation of the occurrence of AGPs in hepatics. Their presence can be detected in tissues as well as tissue extracts by means of Yariv 0-glucosyl reagent, an artificial antigen which specifically binds, precip- itates, and stains AGPs red-orange. We used this reagent to detect AGPs associated with the cell walls of eight species of hepatics, Haplomitrium mnioides (Lindb.) Schust., Scapania nemorosa (L.) Dum., Nowellia curvifolia (Dicks.) Mitt., Jungermannia leiantha Grolle, Gymnocolea inflata (Huds.) Dum., Fossombronia foveolata Lindb., Riccardia sinuata (Dicks.) Trevis, and Riccia duplex Lorbeer. Since these species represent both subclasses, as well asfour ofseven orders, ofthe Hepaticae, these results suggest that AGPs may be regularly occurring components of liverwort cell walls. Whether AGPs function in morphoregulation remains to be determined. Arabinogalactan proteins (AGPs) are a class of hydroxyproline-containing glycoproteins. Clarke et al. (1978) found them to be present in representa- tives of all phyla of photosynthetic land plants (em- bryophytes) they tested, including Lunularia cru- ciata (L.) Dum. among the Bryophyta. Our interest in AGPs derives mainly from the fact that AGPs typically contain high levels of hydroxyproline in their protein moiety (Fincher et al. 1983). We have accumulated evidence that some hydroxyproline- containing protein(s) (hyp-protein) has a morpho- regulatory function in the Hepaticae. Specifically, we have found that inhibition of hyp-protein syn-

Effects of Methylobacterium spp. strains on rice Oryza sativa L. callus induction, plantlet regeneration, and seedlings growth in vitro1

Abstract Maliti, C. M. (Department of Biology & Med. Lab. Technology, Bronx Comm. College, CUNY, University Avenue & West 181 Street, Bronx, NY 10453), D. V. Basile (Department of Biological Sciences, Lehman College, CUNY, Bronx, NY 10468), and W. A. Corpe (Department of Biological Sciences, Columbia University, New York, NY 10027). Effects of methylobacterium spp. strains on rice Oryza sativa callus induction, plantlet regeneration and seedlings growth in vitro. J. Torrey Bot. Soc. 132: 355–367. 2005.—Leaf surfaces of higher plants are colonized by diverse microorganisms. In the present study we report the isolation two strains of pink-pigmented methylotrophic bacteria (PPFM) designated Q4 & Q5. Both strains significantly stimulated growth and development of two rice cultivars, Japonica cv. CR76 and Indica cv. A301, cultured axenically on MS or B5 medium. Co-incubation of strains Q4 and Q5 with embryo-derived rice calli on B5 medium-devoid of exogenous phytohormones resulted in continued callus growth but inhibited plantlet regeneration. These results mimicked those produced by B5 medium supplemented with auxin and cytokinin. Co-culture of Q4 & Q5 as cobionts with 5 day old Japonica and Indica seedlings resulted in a significant (P ≤ 0.05) stimulation of four growth parameters; root development, stem growth, leaf development and biomass productivity, measured in 9 day growth period. The results of the in vitro experiments indicate that strain Q4 and /or Q5 inhabiting rice plant surfaces under cultivation could significantly stimulate the plants growth and development.

New Procedures of Bryophyte Culture Which Permit Alteration of the Culture Medium during the Life Cycle

The essentials of the procedures described are the use of serum bottle stoppers on culture vessels, hypodermic syringes for through-the-stopper inoculations, acid-treated sand 1 I wish to express my appreciation to Dr. Edwin B. Matzke for his advice and help with this work, much of which was facilitated by National Science Foundation Summer Fellowships for Graduate Teaching Assistants during 1960, 1961, 1962. 2 Dept. of Botany, Columbia University, New York, New York 10027. This content downloaded from 207.46.13.51 on Sun, 19 Jun 2016 05:55:59 UTC All use subject to http://about.jstor.org/terms 142 THE BRYOLOGIST [Volume 67 as a substratum, and a quantitative dilution method for the addition of chemical substances. Some of the advantages of each aspect of the procedures are pointed out. The adaptability of some of the methods for the culture of other organisms is suggested. Although the bryophytes are well suited for developmental studies, procedures for their aseptic culture, so important to controlled experimentation, are somewhat less suited. The methods commonly used are designed primarily for the short-term culture of microorganisms and consequently are of only limited use in studies carried out over longer periods. This limitation was previously pointed out by Geldreich (1948) and Schelpe (1953), both of whom devised other ways of circumventing the difficulties. In his aseptic culture of Leucolejeunea, Geldreich used glass cloth to serve both as a support for the spores and as a wick to keep the sporelings supplied with nutrients. Schelpe cultured a number of bryophytes on acid-washed sand in such a way as to minimize contamination but not eliminate it. Both investigators prevented desiccation by adding water or mineral solution to their cultures as needed. In experiments designed to determine the effects of several amino acids on growth and the differentiation of gemmae in Scapania nemorosa (L.) Dum., some new experimental procedures were developed. These procedures permitted the amino acids to be added to the substratum, on which gametophytes were already growing, at three different stages of ontogeny. Although these techniques were developed for a specific type of experiment, they may offer a number of general advantages. The purpose of this paper is to present the features of these procedures that may be of more general value. Specific examples of each aspect of the technique are drawn from an experiment in which they were successfully employed. The results of this experiment will be considered in a subsequent publication. MATERIALS AND METHODS Culture Vessels. The culture vessels used were 60 ml serum bottles with special rubber stoppers, each pierced with a hypodermic needle plugged with cotton. The cotton-plugged needles remained in the stoppers to provide for gaseous interchange and to allow for the equilibration of pressure when materials were injected into (or withdrawn from) a culture bottle by means of a second needle attached to a syringe (Fig. 2). The special rubber stoppers provide for through-the-stopper inoculation of material with a minimum of exposure to airborne contaminants. Cultures have been maintained in serum bottles stoppered in this manner for over a year without perceptible desiccation. Any culture vessel that will accept the stoppers (or, for which this kind of a stopper can be manufactured) will serve as well or, in some cases perhaps, better than serum bottles (Fig. 1). Vessels with This content downloaded from 207.46.13.51 on Sun, 19 Jun 2016 05:55:59 UTC All use subject to http://about.jstor.org/terms 19641 BASILE: BRYOPHYTE CULTURE 143 optically flat surfaces such as those shown in Fig. 1 (a and e), are necessary if close microscopical observation is required. Acid-washed Sand. White quartz sand of fairly uniform particle size was prepared by wetting it to excess with an 8:2 mixture of concentrated sulfuric-nitric acids and heating it in order to oxidize any organic material present. After rinsing with distilled water to remove most of the sulfuric-nitric acids, the sand was similarly treated with concentrated hydrochloric acid to remove heavy metal contaminants. The sand was again rinsed with distilled water, this time until no trace of the acid could be detected with Hydrion pH paper. Finally, it was dried in a forced-draft drying oven. In spite of the labor involved in its preparation, sand was still considered the best material to solidify the substratum in cultures in which a controlled modification of the substratum is important. Some advantages in using acid-washed sand are: 1. It permits rapid diffusion of added substances. 2. It allows for controlled moistening of the substratum. 3. It is relatively inert biologically. 4. It provides for aeration from below. 5. It more closely simulates the texture of the natural substrata. 6. It can be handled quantitatively. 7. It is not altered at the higher and lower pH limits at which some bryophytes grow. Alteration of the Substratum. The means by which the alteration of the substratum was effected was simply to inject 1 ml of an amino acid solution at 10 X concentration into each culture vessel. Since the vessels contained 9 ml of mineral medium, the amino acid solution was quantitatively diluted to its desired concentration. This quantitative dilution method can be used to add a variety of nutrients, metabolites, and growth controlling substances to established cultures. Preparation of Inoculum. A suspension of gemmae in a mineral medium was used as inoculum in the experiment with Scapania. This was prepared by flooding a culture (on agar) which was producing gemmae abundantly. The culture was shaken to free the gemmae and the resultant concentrated suspension was drawn into a syringe and injected into a bottle of sterile mineral medium to give a more dilute suspension. Obviously, any kind of propagules that can be drawn into a syringe would be suitable for preparing inocula to be introduced through-the-stopper. A suspension which is to be used to initiate experimental cultures should be checked for possible contamination by incubating several aliquots on complex organic media. When quantitative inoculation is desired, the concentration of the propagules should be determined. In the case of the gemmae suspension mentioned above, this was done by means of a haemacytometer. The basal culture medium should be used to make the suspension of inocula. This content downloaded from 207.46.13.51 on Sun, 19 Jun 2016 05:55:59 UTC All use subject to http://about.jstor.org/terms 144 THE BRYOLOGIST [Volume 67

Patterns in Plant Development.

Preface Acknowledgements 1. Development in the vascular plants 2. Embryogenesis: beginnings of development 3. Analytical and experimental studies of embryo development 4. The structure of the shoot apex 5. Analytical studies of the shoot apex 6. Experimental investigations on the shoot apex 7. Organogenesis in the shoot: leaf origin and position 8. Organogenesis in the shoot: determination of leaves and branches 9. Organogenesis in the shoot: later stages of leaf development 10. Determinate shoots: thorns and flowers 11. The development of the shoot system 12. The root 13. Differentiation of the plant body: the origin of pattern 14. Differentiation of the plant body: the elaboration of pattern 15. Secondary growth: the vascular cambium 16. Seconary growth: experimental studies on the cambium 17. Alternative patterns of development Credits Author index Subject index.