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Biochemical characterization of developmental stages of cycad somatic embryos.

 I. Abstract\Resumen
 II. Introduction
III. Materials and Methods
 IV. Results and Discussion
 V. Acknowledgments
 VI. Literature Cited

II. Introduction

Ceratozamia mexicana is considered to be a species in danger of extinction in its native habitats in Central America. It has been possible to obtain somatic embryos of this species from megagametophyte and zygotic embryos (Chavez et al., 1992a) and from leaves of maturephase plants (Chavez et al., 1992b). Somatic embryogenesis can be utilized for studying growth and development, conservation, and improvement. These results could provide an alternative method for propagating this and other cycad species that are seriously endangered; however, control of the maturation of somatic embryos and the recovery of plants is only beginning to be studied. Consequently, the establishment of plants in soil is rather inefficient and currently limits the best use of this method for propagation and conservation of cycads.

Webb (1983) evaluated the effect of light on zygotic embryos and plants grown in vitro. Under low light conditions, root nodules could be induced in Zamia pumila without affecting growth of the apical meristem. Later, Webb (1984) showed that callus could be initiated from roots of Dioon edule under light conditions. There is little information on growth and development responses of somatic embryos. They have been generally described with reference to changes in their morphology and fresh weight, without examining any of the biochemical parameters associated with development, such as changes in protein content, including peroxidase and invertase activities, and carbohydrate content. Biochemical markers could be useful indicators of normal vegetative development (Borkowska & Opilowska, 1988; Preece & Sutter, 1991; Ziv, 1991; Sanchez et al., 1995).

Increase in peroxidase activity is associated with a decrease in tissue growth due to narrowing of the cell walls. Crossed covalent links among wall polymers are formed due to oxidation of phenolic residues from polysaccharides and structural proteins catalyzed by peroxidase activity. There is little doubt about this, even though they also participate in other functions such as elongation inhibition by AIA catabolism or by oxidation of 1--aminocycle propane 1--carboxilic acid into ethylene (Gaspar et al., 1985). Peroxidase activity ionically united to the wall is accepted as representative of that wall's activity and is specifically related to interruption of cell expansion or elongation (Sanchez et al., 1995).

Sucrose is the most frequently used carbohydrate in tissue culture. A constant exchange of sucrose has been found, even though this is not the immediate source of carbon in cultured tissues. Before being used it has to be hydrolyzed to glucose and fructose. In this process of exchange and utilization, invertase plays an important role (Borkowska & Kubik, 1990).

In this study, the developmental morphology of four distinct stages of C. mexicana somatic embryos is described and correlated with peroxidase and invertase activity. The activities of these enzymes appear to be useful metabolic markers for maturation under different light conditions.

III. Materials and Methods

Somatic embryos of Ceratozamia mexicana representing four different developmental stages were isolated from embryogenic cultures that had been derived from explanted zygotic embryos. The induction medium consisted of major salts (Gamborg et al., 1968), minor salts (Murashige & Skoog, 1962), and organic components and was supplemented with 100 mg/liter arginine, 100 mg/liter asparagine, 400 mg/liter glutamine, 60 g/liter sucrose, 0-0.1 mg/liter 2,4-D, 1-2 mg/liter kinetin, and 8.0 g/liter agar. Cultures were maintained on the same medium formulation without plant-growth regulators. The cultures were grown in baby-food jars with polypropylene closures that were sealed with Saran. Stage 1 somatic embryos (F1) were collected in two amounts (0.5 [cm.sup.3]) from each culture container. A single somatic embryo from each of stages 2 through 4 (F2-F4) was collected from each container. There were 10 replicates for each developmental stage, and the experiment was repeated two times. The experimental treatments (all at 25[degrees]C) were as follows:

1. 1-hour light daily pulses 50 [micro]mol [m.sup.-2][s.sup.-1], for 1 week adding up until reaching 16 hours per day the 16th week

2. 16-hour photoperiod at 25 [micro]mol [m.sup.-2][s.sup.-1]

3. 16-hour photoperiod at 10 [micro]mol [m.sup.-2][s.sup.-1]

4. 16-hour photoperiod at 5 [micro]mol [m.sup.-2][s.sup.-1]

5. Darkness

The cultures were subcultured onto fresh maintenance medium at three-month intervals. After the third subculture, agar was replaced by gellan gum (3.0 g/liter), and the cultures were maintained for two years in order to determine the time required for passage from one developmental stage to the next.

In parallel, biochemical studies were carried out three months after subculture using those cultures grown under 25 and 50 [micro]mol [m.sup.-2][s.sup.-1] light conditions. Soluble proteins and cell-wall traits (Bradford, 1976) in terms of the ionic form and invertase activity (Borkowska & Opilowska, 1988), peroxidase activity (MacAdam et al., 1992) and glucose (Nelson, 1944) were measured in flesh tissue.

IV. Results and Discussion

In all of the treatments, friable embryogenic cultures (F1) were produced that later developed recognizable precotyledonary somatic embryos, each consisting of a densely white meristematic region subtended by a hyaline suspensor (F2). The precotyledonary embryos were ca. 1 cm long and 1-1.5 mm wide. Cotyledonary somatic embryos developed from the meristematic region (F3) were two or three times the length of the precotyledonary embryo. Complete, bipolar somatic embryos (F4) germinated and continued to enlarge.

In general, better somatic embryo development was observed in treatments with 25 and 50 [micro]mol [m.sup.-2][s.sup.-1] photoperiods. Development was asynchronous. The higher light intensity stimulated development to the next stage more rapidly than did low light regimes and darkness (Table I). Secondary embryogenic cultures (F1) having a light brown appearance were induced from older developmental stages(>F2) and provided a continuous source of somatic embryos.

There was a significantly greater increase in the fresh weight of somatic embryos in stages F2 and F4 that were grown under a 25 [micro]mol [m.sup.-2][s.sup.-1] photoperiod. With a 50 [micro]mol [m.sup.2][s.sup.-1] photoperiod, the highest weight increase occurred in somatic embryos at stages F1 and F4 (Fig. 1). For both light conditions, the greatest weight gain occurred in stages F2-F4 under low light conditions (25 [micro]mol [m.sup.-2][s.sup.-1]) and appeared to be associated with a high incidence of hyperhydricity, especially with stage 2 somatic embryos. There was also a marked elongation of somatic embryos representing the same stages during the three-month incubation period.


Peroxidase activity was observed in somatic embryos at all stages but appeared to be lower in cultures that were incubated under low light intensity. The activity of peroxidase was greatest in somatic embryos at stages F3 and F4. We also observed strong peroxidase activity in cultures representing stage 1 when grown at 50 [micro]mol [m.sup.-2][s.sup.-1], although this decreased in stage 2 (0.07 O.D./g F.wt./min). In stage F3 and F4 somatic embryos we did not observe activity of the soluble enzyme and only registered peroxidase activity as it occurred in the ionic form. This increase in enzyme activity could have been associated with a slower rate of growth of the cultures, which did not occur in stages 1 and 2 under low light conditions (Fig. 2).


Although development from the proembryonic to precotyledonary stage occurred more slowly than did other transitions, the precotyledonary somatic embryos that developed under high light conditions showed less hyperhydricity and possibly better adaptation to soil conditions. Vigorous growth was observed with higher lignification and a strong transition to autotrophy.

Pinus pinaster cotyledons also showed a reverse relationship between increased activity of peroxidase and slower rate of elongation of the precotyledonary embryo (Sanchez et al., 1995). The activity of peroxidase was less in elongation regions than in normal growth zones in Festuca arundinacea (MacAdam et al., 1992) and Linum usitatissimum (McDougall, 1992).

In Phoenix dactylifera evolution of peroxidase activity strongly reflected morphological changes observed during culture, with an increase in its activity associated with a decrease in growth by elongation. The conclusion is that studying the evolution of peroxidase activity during culture can help us understand the physiological processes that occur (Booij et al., 1993).

In Syngonium podophyllum plants cultivated in vitro, a very low peroxidase activity was found compared with those cultivated on soil. An increased peroxidase activity of nearly 300% was determined for plantlets cultured in vitro that were transferred to soil. From that came the hypothesis that the culture atmosphere has an inhibitory effect on peroxidase activity and almost certainly on other enzymes that participate in the lignification of plant tissues (Salame & Zieslin, 1994). It is also possible that the low rate of lignification can affect the vulnerability of in vitro plants transferred to soil (Preece & Sutter, 1991; Ziv, 1991).

Invertase activity was less in stage F2 (0.7 mg glucose/g F.wt./min) and could have been due to the adaptation of somatic embryos to lower light conditions (25 [micro]mol [m.sup.-2][s.sup.-1]) at this stage. Invertase activity in the other developmental stages remained constant (Fig. 3).


Stage F2 somatic embryos that developed under these conditions had a metabolism characterized by a low level of invertase activity and a high rate of utilization of glucose (in comparison with stage F1). This could have been due to elevated respiration together with rapid elongation. On the other hand, the activity of this enzyme under high illumination increased strongly in stage F2 and later decreased in stages F3 and 4 in relation to the gradual increase in autotrophy. The color of the somatic embryos changed from white to green, and the internal concentration of glucose increased (Fig. 4). High illumination caused an increase of this activity, resulting in the same levels of internal glucose that were observed in stage F1 embryos.


The activity of this enzyme is essential for growth and results in the metabolism of sugar for division and cell expansion (Borkowska & Opilowska, 1988). There is a strong correlation between accumulation of reducing sugars and the expansion of cotyledons under light conditions of Raphanus sativus (Howard & Witham, 1983). Cotyledons that grow under light conditions can utilize sucrose as a source of carbon, and the accumulation of sugar depends directly on invertase activity. This corresponds with the opposite in our study, in which high light conditions during embryo development resulted in a significant increase in invertase activity.

Invertase activity in Prunus cerasus was dependent on the sucrose in the environment. Thus the activity of this enzyme can be an indicator of the degree of tissue heterotrophy (Borkowska & Szczerba, 1991).

In Spathiphyllum plantlets a direct relationship between the presence of sucrose in the environment and acid invertase activity among other enzymes was found. Also, we observed rapid C[O.sub.2] fixation during the first days after transfer to soil and a rapid decrease of acid invertase activity from the moment of transfer. We concluded that plantlet photosynthetic activity at the moment of transfer is of secondary importance, the main requirement being the nutrient reserve in the form of sucrose (Van Huylenbroeck & Debergh, 1996). In this way the behavior of the invertase activity should be the determinant for the accumulation of glucose, fructose, and sucrose in tissues cultured in vitro, both autotrophic and semiautotrophic.

The growth of somatic embryos was more rapid under high light conditions (50 [micro]mol [m.sup.-2][s.sup.-1]) than under the other growth conditions explored in this study and caused a better adaptation later on, when somatic embryos were transferred to soil. These somatic embryos were more lignified and appeared to be autotrophic.

These results contribute to the study and conservation of cycads, even if somatic embryo maturation and plantlet establishment on soil are a limiting factor in the whole process of micropropagation of this seriously endangered species.

V. Acknowledgments

The authors are grateful for the support of their respective institutions and for the initial inspiration for this and other studies provided by Dr. Knut Norstog.

VI. Literature Cited

Booij, I., S. Monfort & J. J. Macheix. 1993. Relationships between peroxidases and budding in date palm tissues cultured in vitro. PI. Cell Tissue Organ Cult. 35: 165-171.

Borkowska, B. & M. Kubik. 1990. Utilization and accumulation of [sup.14]C--sucrose in sour cherry shoots rooted in vitro. Sci. Hurt. 44: 261-267.

-- & M. Opilowska. 1988. Influence of BA and other cytokinins on proliferation and metabolic status of sour cherry cultures cultivated in vitro. Fruit Sci. Rep. 15(4}: 147-156.

-- & J. Szezerba. 1991. influence of different carbon sources on invertase activity and growth of sour cherry (Prunus cerasus L.) shoot cultures. J. Exp. But. 42:911-915.

Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein-dye binding. Ann. Biochem. 72: 248-254.

Chavez, V. M., R. E. Litz & K. Norstug. 1992a. In vitro morphogenesis of Ceratozamia hildae and C. mexicana from megagametophytes and zygotic embryos. P1. Cell Tiss. Org. Cult. 30: 93-98.

--, --, P. A. Moon & K. Norstog. 1992b. Somatic embryogenesis from leaf callus of a mature gymnosperm Ceratozamia mexicana var. robusta (Miq.) Dyer (Cycadales). In Vitro Cell. Devel. Biol. 28P: 59-63.

Gamborg, O. L., R. A. Miller & K. Ojima. 1968. Plant cell cultures, I. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50:151-158.

Gaspar, T., C. Penel, F. J. Castillo & H. Greppin. 1985. A two-step control of basic and acidic peroxidases and its significance for growth and development. Physiol. P1. 64:418-423.

Howard, H. F. & F. H. Witham. 1983. Invertase activity and the kinetin-stimulated enlargement of detached radish cotyledons. Pl. Physiol. 73: 304-308.

MacAdam, J. W., R. Sharp & C. J. Nelson. 1992. Peroxidase activity in the leaf elongation zone of tall fescue. Pl. Physiol. 99: 879-885.

McDougall, G. J. 1992. Changes in cell wall-associated peroxidases during the lignification of flax fibres. Phytochemistry 31: 3385-3389.

Murashige, T. & F. Skoog. 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Pl. 15: 473-497.

Nelson, N. 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153:375-380.

Preece, J. E. & E. G. Sutter. 1991. Acclimatization of micropropagated plants to the greenhouse and field. Pp. 23-33 in P. C. Debergh & R. H. Zimmerman (eds.), Micropropagation: Technology and application. Kluwer Academic Publishers, Dordrecht, Netherlands.

Salame, N. & N. Zieslin. 1994. Peroxidase activity in leaves of Syngonium podophyllum following transition from in vitro to ex vitro conditions. Biol. Pl. 36(4): 619-622.

Sanchez, M., G. Revilla & I. Zarra. 1995. Changes in peroxidase activity associated with cell walls during pine hypocotyl growth. Ann. Bot. 75: 415-419.

Van Huylenbroeck, J. M. & P. C. Debergh. 1996. Impact of sugar concentration in vitro on photosynthesis and carbon metabolism during ex vitro acclimatization of Spathiphyllum plantlets. Physiol. Pl. 96: 298-304.

Webb, D. T. 1983. Developmental anatomy of light-induced root nodulation by Zamia pumila L. seedlings in sterile culture. Amer. J. Bot. 70(8): 1109-1117.

--. 1984. Developmental anatomy and histochemistry of light-induced callus formation by Dioon edule (Zamiaceae) seedling roots in vitro. Amer. J. Bot. 71(l): 65-68.

Ziv, M. 1991. Vitrification: Morphological and physiological disorders of in vitro plants. Pp. in E C. Debergh & R. H. Zimmerman (eds.), Micropropagation: Technology and application. Kluwer Academic Publishers, Dordrecht, Netherlands.

I. VARGAs--LUNA, (1) G. ORTIZ--MONTIEL, (2) V. M. CHAVEZ, (1) R. E. LITZ, (3) AND P. A. MOON (3)

(1) Jardin Botanico, Instituto de Biologia Universidad Nacional Autonoma de Mexico 04510 Mexico DF, Mexico

(2) ENEP-Iztacala Universidad National Autonoma de Mexico, 04510 Mexico DF, Mexico

(3) Tropical Research and Education Center University of Florida Homestead FL 33031-3314, U.S.A.
Table I
Time for somatic embryos to develop from one stage to the next

Stage Light intensity Time

 1 50 [micro] mol [m.sup.-2] [s.sup.-1] 2.5-3.0 months
 2 25 [micro] mol [m.sup.-2] [s.sup.-1] 3.0-4.0 months
 3 10 [micro] mol [m.sup.-2] [s.sup.-1] 3.5-6.0 months
 4 5 [micro] mol [m.sup.-2] [s.sup.-1] 3.5-6.0 months
 5 Darkness 3.5-6.0 months
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Article Details
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Author:Vargas-Luna, I.; Ortiz-Montiel, G.; Chavez, V.M.; Litz, R.E.; Moon, P.A.
Publication:The Botanical Review
Geographic Code:20CEN
Date:Jan 1, 2004
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