Annals of Botany 89: 645-648, 2002
© 2002 Annals of Botany Company
Effect of Medium Salt Concentration on Differentiation and Maturation of Somatic Embryos of Cassava (Manihot esculenta Crantz)
1School of Animal, Plant and Environmental Sciences, Private Bag 3, University of the Witwatersrand, PO 2050, Johannesburg, South Africa and 2School of Molecular and Cellular Biology, Private Bag 3, University of the Witwatersrand, PO 2050, Johannesburg, South Africa
* For correspondence. E-mail dave{at}gecko.biol.wits.ac.za
Received: 19 April 2001; Returned for revision: 9 January 2002; Accepted: 27 January 2002.
ABSTRACT
Culture of cassava somatic embryos on media with an altered macro- and micro-nutrient salt concentration affected embryo development and germination capability. In the tests, quarter-, half-, full- or double-strength Murashige and Skoog (MS) media were compared. The maximum number of somatic embryos differentiated from a proliferative nodular embryogenic callus (NEC) on either half- or full-strength MS medium, and the greatest numbers of cotyledonary stage embryos were formed on full-strength MS medium. Developed somatic embryos were then desiccated above a saturated K2SO4 solution for 10 d. After transfer to germination medium, embryos that had developed on half- and full-strength MS medium yielded 8·3 and 8·6 germinants g–1 NEC tissue, respectively. For this important but often disregarded culture factor, either half- or full-strength MS medium is recommended for both the differentiation and development of cassava somatic embryos that are capable of germination.
Key words: Cassava, Manihot esculenta, secondary somatic embryogenesis, salt concentration, nodular embryogenic callus.
INTRODUCTION
A frequent in vitro culture manipulation for cassava (Manihot esculenta Crantz) involves culture on a standard medium such as Murashige and Skoog (1962) (MS medium), but with altered macronutrient and/or micronutrient concentrations. This manipulation was initially restricted to embryo culture and nodal micropropagation, but it has recently been extended to somatic embryogenesis. Propagation on half-strength MS medium was used for nodal culture and multiple shoot induction by Smith et al. (1986), while one-third strength MS medium was used for rooting through to hardening stages for culture from meristem explants (Roca, 1984). A medium low in salts was similarly used for the recovery of plants following culture of zygotic embryos, with concentrations varying from one-third-strength MS (Fregene et al., 1999) to half-strength MS (Biggs et al., 1986). For the germination of somatic embryos, Mathews et al. (1993) used MS medium with major salts at half-strength.
It had previously been observed that the concentration of inorganic constituents in the MS medium was suboptimal for cassava growth (Meyer and van Staden, 1986). Specific research on somatic embryogenesis has yielded contradictory information in this regard after some researchers tested alternative media and/or MS medium at different strengths and with alternative nitrogen sources. Taylor et al. (1996) observed that half-strength MS medium was superior to full-strength MS medium for the induction of a friable embryogenic callus, although the medium of Gresshoff and Doy (1974) was most effective for the production of this tissue. The viability of meristems excised from expanded axillary buds of cassava on media containing half and quarter of the concentration of MS salts was also enhanced relative to that of buds on full-strength MS medium (Konan et al., 1997). In contrast, more primary somatic embryos developed on full-strength MS medium than in half-strength and double-strength MS, although the regenerative quality of such embryos was not described (Konan et al., 1994).
Due to the importance of this culture factor, a study was conducted to determine the optimal MS medium concentration for differentiation and development of secondary cassava somatic embryos. This study was also extended to ascertain whether the use of this medium had a delayed influence on regenerative ability following desiccation.
MATERIALS AND METHODS
Source material and culture conditions
All embryos used in this study were formed by indirect secondary embryogenesis from a nodular embryogenic callus (NEC) tissue as described in Groll et al. (2001). Four independently obtained NEC tissue lines from cultivar MCol 22 (provided by CIAT, Colombia) were used as starting material. Culture plates were maintained in a controlled environment (Conviron CMP3244) chamber at a temperature of 28 ± 2 °C under a 16 h photoperiod and a photosynthetic photon flux of 100 µmol m–2 s–1 provided by cool white fluorescent lamps.
Developmental media
Samples of 100 mg (fresh mass) fragmented NEC tissue were used to innoculate 25 mm sterile Petri plates containing a medium consisting of either quarter-, half-, full- or double-strength MS micro- and macro-nutrients and supplemented with normal-strength MS vitamins, 2 g l–1 Gelrite and 20 g l–1 sucrose. Thirty replicates were used per treatment. Tissue samples were maintained above the medium on an autoclaveable 225 µm (pore diameter) nylon mesh to facilitate tissue transfer during subculture. Media were adjusted to pH 5·7 prior to autoclaving at 121 °C for 20 min. Tissue was maintained on this medium for 18 d prior to desiccation, with tissue being subcultured onto fresh media once during this period (after 7 d of culture). Counts of embryos at the pre-torpedo, torpedo and cotyledonary stages of development were made after 14 d of culture.
Desiccation
Developed embryos were removed from attached callus and were transferred, in groups of three, to a sterile plastic float above a saturated solution of K2SO4 for 10 d at a temperature of 25 ± 0·5 °C (relative humidity 97·5 %; Winston and Bates, 1960). No selection was made for the stage of development of individual embryos. Each individual replicate was desiccated separately in its own sterile jar (100 ml volume) containing a single plastic float above the saturated salt solution (40 ml).
Water content determination
Gravimetric determinations of tissue water content were made at 80 °C from embryos post-desiccation. Measurements were expressed as g H2O g–1 (fresh mass basis), with 35–40 replicates (each comprising three embryos) being used for water content determinations. Experimental repeats were used to allow for water content determinations that were destructive in nature.
Germination
To determine germination ability, desiccated embryos that were not used for water content determination were re-imbibed directly on germination medium consisting of full MS salts and vitamins supplemented with 2 g l–1 Gelrite, 20 g l–1 sucrose and 0·1 mg l–1 6-benzylaminopurine. Numbers of germinants were counted 30 d after plating on germination medium. Germination was defined by the production of new leaves that were not present prior to desiccation, as well as elongation of the hypocotylar portion.
RESULTS AND DISCUSSION
Proliferation of the NEC tissue ceased upon transfer to phytohormone-free medium and discernable embryogenic structures were observed as early as 4–5 d after transfer to the developmental media. The sequence of development was similar to that described by Raemakers et al. (1993) with embryos initially appearing as a small ring of green spike-like structures and then progressing through a torpedo stage of development to a cotyledonary stage.
Significantly higher numbers of embryos differentiated when cultured on half- and full-strength MS media (Table 1). However, only 20·5 % of the 2150 embryos that developed in total could be classed as cotyledonary. The full-strength MS medium yielded the maximum number of cotyledonary-stage embryos at 62·3 g–1 NEC (24·9 %).
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The appearance of developed somatic embryos differed greatly between treatments. Embryos differentiating on quarter-strength MS medium were reduced in size and were pale yellow in colour. Embryos from the double-strength MS medium showed more cotyledonary abnormalities including excessive enlargement of cotyledons. Visually, embryos from both of these treatments could be classed as poor relative to embryos from the half- and full-strength MS treatments. This was reflected in a significantly greater fresh mass of embryos from the half- and full-strength MS treatments (20·3 and 17·6 mg, respectively) (Table 2).
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Desiccation reduced moisture contents from 96–98 % prior to desiccation to 88–93 % post desiccation (Table 2). The water content of pre-desiccated somatic embryos decreased with increasing salt concentration in the medium.
Greatest dry mass accumulation occurred for the full-strength MS treatment with the mean dry weight after desiccation being 0·42 ± 0·04 mg per sample (0·14 mg per embryo). The method used for desiccation was reliable and consistent among samples and within treatments studied, particularly when measured in terms of percentage loss of initial fresh mass, which decreased linearly as salt concentration decreased (Table 2).
The high moisture contents (96–98 %) before desiccation differed considerably from published data for other species, and consequently also affected water content after desiccation. For white spruce, Attree et al. (1991) observed moisture contents in the range of 40–60 % in embryos that had been exposed to a suitable maturation treatment. Similarly, for soybean, Buchheim et al. (1989) observed that the water content of somatic embryos after a shorter maturation treatment was approx. 85 %, falling to 50 % after a longer period of maturation. Differences in water content for the cassava somatic embryos may be attributed to the fact that these embryos had not undergone a suitable maturation treatment prior to desiccation and, as a consequence, had accumulated relatively little dry mass (Table 2). For white spruce, culture on media supplemented with abscisic acid (ABA) and a non-plasmolysing moisture stress effected by polyethylene glycol are congruent with increasing embryo dry mass accumulation.
Highest numbers of germinants were observed for the half- and full-strength MS treatments (8·3 and 8·6 germinants g–1 NEC tissue, respectively) (Table 2). Germination was expressed in terms of the number of germinants per unit mass proliferative embryogenic tissue in this study, since this provided a measure of the absolute regenerative capability of the tissue. To measure germination in this way it was necessary to desiccate all differentiated embryos formed on the developmental media. Schultheis et al. (1990) observed that sweet potato somatic embryos at the mature stages are better suited for plantlet formation. Selection of prime cotyledonary stage embryos on the basis of normality of appearance and existence of a clear shoot meristem could be used to further enhance the germination frequency.
This study was conducted to determine the optimal medium salt concentration for the development of somatic embryos prior to the introduction of culture manipulations designed to enhance embryo maturation. Such culture manipulations may include culture of differentiated embryos on media with increased sucrose content, ABA supplementation of the maturation medium or the introduction of osmotica such as polyethylene glycol. Since culture on media with increased salt concentration increases the osmotic concentration of the medium, optimization of the salt concentration prior to the introduction of maturation treatments was considered to be a priority, especially as this factor has been demonstrated to be of importance in related cassava research (Konan et al., 1994). It is worth noting that this study does not rule out the possibility that the effect of a combination of medium salt concentration together with addition of external plant growth regulators to the medium may prove to be more significant than the effect of medium salt concentration alone.
As demonstrated by the relatively low dry matter accumulation and high water content of somatic embryos that develop without a maturation treatment, introduction of such a maturation step to the protocol, as applied here, may be used to develop a system for the production of desiccated cassava somatic embryos with adequate dry matter accumulation. The moisture content measurements for secondary somatic embryos observed here are surprising when compared with a moisture content for whole cassava seed of approx. 7·9 % (Ellis et al., 1981).
In summary, there was no significant difference between half- and full-strength MS media for the production of differentiated cassava somatic embryos as judged by number of embryos and/or germination capability. However, embryos differentiating on full-strength MS medium showed production of a greater number of cotyledonary-stage somatic embryos, with increased dry matter accumulation.
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