SUITABILITY OF SOME AGAR SUBSTITUTES FOR THE PROPAGATION OF SPATHIPHYLLUM AND ANTHURIUM BY TISSUE CULTURE TECHNIQUE IN MULTIPLICATION STAGE

SUITABILITY OF SOME AGAR SUBSTITUTES FOR THE PROPAGATION OF SPATHIPHYLLUM AND ANTHURIUM BY TISSUE CULTURE TECHNIQUE IN MULTIPLICATION STAGE

F. M. Saadawy *; M. A. Darwish **; H. M.Fahmy ** and H.H.M Hassan *

* Ornamental Plants and Landscape Design Research Dept., Horticulture Research Institute, Agriculture Research Center, Giza, Egypt.

** Department of Ornamental Plants, Faculty of Agriculture, Cairo University, Giza, Egypt.

ABSTRACT

This study was carried out in a trial to find out a cheaper substitute for agar. Different substrates were tested as support matriceswith MS solution and compared with agar-gelled MS medium. Aseptic explants of two aroids, Spathiphyllum wallisii and Anthurium scherzerianum were cultured on these media under aseptic conditions.

The highest records of total number of shoots, shoot length, total number of leaves, diameter of vegetative growth and total fresh weight of Spathiphyllum were observed in shoots grown on agar medium. Results of Anthurium shoots grown on agar medium did not differ significantly from the highest levels of shoot length and total fresh weight. Agar also achieved the second rank in total number of shoots, total number of leaves and the diameter of vegetative growth of Anthurium shoots.

Length of Spathiphyllum and Anthurium shoots grown on cotton medium was not significantly different from the highest level. Cotton also achieved the second rank in total number of shoots, total number of leaves, diameter of vegetative growth and total fresh weight of Spathiphyllum and Anthurium shoots.

In case of Spathiphyllum, perlite medium achieved the second rank in total fresh weight, and was not significantly lower than this category in total number of leaves. Records of Spathiphyllum shoots grown on perlite were lowered to the third rank in total number of shoots, shoot length and diameter of vegetative growth. On the other hand, the highest significant levels in total number of shoots, shoot length, total number of leaves, diameter of vegetative growth and total fresh weight of Anthurium were observed in shoots grown on perlite. 

Value of the total fresh weight of Spathiphyllum shoots grown on bagasse medium significantly occupied the second rank in a descending order compared to the corresponding character in other media. The bagasse medium reached the third category in shoot length and total number of leaves, and the fourth one in total number of shoots and diameter of vegetative growth of Spathiphyllum. In case of Anthurium, values of all characters measured of shoots grown in the bagasse medium significantly reached the third rank in a descending order.

The longest shoots were those grown on straws of rice or wheat in case of Spathiphyllum, and on rice straw in case of Anthurium.

Parameters of the photosynthetic pigments, i.e. chlorophyll a, chlorophyll b, total chlorophyll and carotenoids, were not influenced significantly by media substrate in case of Spathiphyllum. On the contrary, the highest significant values of these characters were observed in Anthurium shoots grown on bagasse medium, followed without any significant difference by those grown on the cotton one. Corresponding values of Anthurium shoots grown on perlite medium were not significantly different from those grown on agar medium. However, values of the two later media were significantly lower than those of the two former ones.

The use of cheaper substrates such as cotton, perlite or bagasse could be recommended as a replacement for agar, especially in commercial tissue culture labs.

Key words: Spathiphyllum, Anthurium, tissue culture, substrate, agar, cotton, perlite, bagasse, rice straw, wheat straw, rice hull, bran, sand.

INTRODUCTION

Agar is an unbranched polysaccharide complex obtained through bleaching and hot water extraction of agarocytes from the red algae (Rhodophyceae), found in the Pacific and Indian Oceans, the Sea of Japan and off the coasts of Spain and Mexico, with Spain being the world's largest supplier. Chemically, agar is a polymer made up of subunits of the sugar galactose. Agar is a heterogeneous mixture composed of about 70% agarose and 30% agaropectin. Agarose is a neutral gelling fraction, while agaropectin is a non-gelling fraction (Merck Index, 1996).

Agar has remained the most frequently used gelling agent for microbial and plant tissue culture media since its introduction, more than a hundred years ago by Robert Koch (Pelczar et al., 1986). The properties of agar, which make it the gelling agent of choice, are stability, high clarity and resistance to metabolism during culture. However, some investigators have raised doubts about its biological inertness and reported about its cytotoxic effects in some cases. (Jain and Babbar, 2005). Due to these reasons and the high price of tissue-culture-grade agar, attempts were initiated to identify suitable alternatives.

Other gelling agents include (in alphabet order):

1 - Agarose: Agarose is a component and purification product of agar. It is superior to agar in gelling ability and transparency. However, it cannot be used for routine experiments and in plant tissue culture industry because of its high cost (Johansson, 1988).

2 - Agargel: Agargel is a blend of agar and phytagel. It is cheaper than agar, and is superior to phytagel in applications where vitrification is a problem in plant tissue cultures. Agargel produces a semiclear gel which allows for better detection of contamination (Sigma Chemical Co., 1996).

3 - Algin, Sodium alginate: Algin or sodium alginate is prepared from brown algae. Chemically it is a linear unbranched, hydrophilic, colloidal, polyuronic acid composed primarily of anhydro-β-D-mannuronic acid residues. Alginic acid has been used as a physical support similar to agarose and to prepare beads into which immobilized suspension cells and protoplasts are embedded. Aqueous solutions of alginate form a gel at room temperature in the presence of certain cations, especially calcium. This gel matrix can be reliquified by adding chelating agents (e.g. citrate). Alginates are thermally stable cold setting gelling agents; gelling at far lower concentrations than gelatin. Such gels can be heat treated without melting, although they may eventually degrade, (Larkin, 1988).

4- Carrageenan, Kappa carrageenan: Carrageenan is a collective term for polysaccharides prepared by alkaline extraction (and modification) from red seaweed (Rhodophycae), mostly of genus Chondrus, Eucheuma, Gigartina and Iridaea. Different seaweeds produce different carrageenans, (Chaplin 2008). (McConnell and Tomomitsu, 1983) reported that there was no significant difference in shoot length of the orchid Dendrobium seedlings grown on agar or on 0.4-1.0% carrageenan. Although the pH of carrageenan media was slightly higher than that of agar, carrageenan appeared to be an acceptable substitute for agar when growing Dendrobium seedlings.

5 – Ficoll: Ficoll is a neutral, highly branched, high-mass, hydrophilic polysaccharide which dissolves readily in aqueous solutions. It is prepared by reaction of the polysaccharide with epichlorohydrin. Ficoll does not form a gel, but makes the medium viscous causing floatation of explants. However, at the used concentration, the cost per litre of medium is higher than even agarose (Kao, 1981).

6 – Gelatin: Gelatin is a protein produced by partial hydrolysis of collagen extracted from the bones, connective tissues, organs, and some intestines of animals such as the domesticated cattle, and horses. (Ward and Courts, 1977)

7 - Gellan gum (gelrite): Gellan gum (gelrite) is a water soluble polysaccharide produced by Sphingomonas elodea (previously called Pseudomonas elodea), a bacterium. It is used primarily as an alternative to agar as a gelling agent. In spite of the many desirable properties of gelrite, it causes hyperhydricity in some cases. However, though not a perfect replacement for agar, it has found wide acceptance (Eyre and Caswell, 1991 and Pasqualetto et al., 1988). It needs only approximately half the amount of gellan gum as agar to reach an equivalent gel strength. Gellan gum (gelrite) has been successfully used, but like agar, it is expensive, (Chaplin 2008). 

8 - Guar gum: Guar gum is isolated from the endosperms of guar (Cyamopsis tetragonoloba, Fabaceae). It has been successfully used as an exclusive gelling agent for in vitro multiplication of an orchid, Dendrobium chrysotoxum from leaf and protocorm explants. However, media gelled with guar gum require quick adjustment of pH and dispensing because of their higher gelling point. As cost of guar gum per litre of the medium is about 2.5–13 times less than different brands of agar, it is a highly cost-effective gelling agent, which could be used for reducing the cost of in vitro-propagated orchid plants, (Jain and Babbar, 2005).

9 - Gum Katira: The successful use of Gum Katira (exudated from the bark of Cochlospermum religiosum, Cochlospermaceae) as exclusive gelling agent for eliciting morphogenic responses as varied as caulogenesis, rhizogenesis, androgenesis, and embryogenesis in some model systems was reported by Babbar and Jain (1998) and Jain and Babbar (2002). Gum Katira, like ficoll, does not form a firm gel at 3%, but the explants remain on the surface if left undisturbed. In transparency, Gum Katira-gelled medium is comparable to the liquid medium. Therefore, it can be an excellent gelling agent for experiments requiring regular observations of cells, tissues or organs growing inside the medium (Jain and Babbar, 2002).

10 – Isubgol: Isubgol is colloidal mucilage from the husk of Plantago ovata seeds. It is an inexpensive gelling agent for plant tissue cultures. Bhattacharya et al. (1994) reported that growth of chrysanthemum (Dendranthema grandiflora) tissues on 2-3% isubgol-solidified medium was about 2.2 times that on agar-solidified medium. Babbar and Jain (1998) described the use of 3% isubgol-gelled medium for in vitro seed germination, shoot formation and rooting in Syzygium cuminii and for anther culture in Datura innoxia, as compared with the use of 0.9% agar-gelled medium. In each case, the response on isubgol-gelled medium was not statistically different from that on agar-gelled medium, nor did the isubgol appear to have any adverse effects on further development of in vitro-regenerated plantlets. Irrespective of its higher melting point (70.6°C), which necessitates quick adjustment of pH and quick dispensing, isubgol is a cost-effective gelling agent with desirable properties (Babbar and Jain, 1998 and Jain and Babbar, 2002). Jain and Babbar (2005) reported that isubgol has been successfully used as an exclusive gelling agent for in vitro multiplication of an orchid, Dendrobium chrysotoxum from leaf and protocorm explants. They also stated that as cost of isubgol per litre of the medium is about 2.5–13 times less than different brands of agar, it is a highly cost-effective gelling agent, which could be used for reducing the cost of in vitro-propagated orchids plants.

11 - Phytagel: Phytagel is an agar substitute produced from a bacterial substrate composed of glucuronic acid, rhamnose and glucose. It produces a clear, colorless, high strength gel which aids in detection of microbial contamination. Phytagel provides an economical alternative to agar as a gelling agent, (Sigma Chemical Co., 1996). Arregui et al. (2003) compared the gelling agent Phytagel  was with Difco Bacto-agar for in vitro tuberization of six potato cultivars. They noticed that tuberization was higher when Phytagel  was used rather than Difco Bacto-agar. Chemical analyses of the gelling agents revealed a higher mineral content and organic impurities in Bacto-agar than in Phytagel.

12 - Starches: Good results have been reported with the use of starches as gelling agents, but starch-gelled media tend to be opaque and soft. Also starches can break down to form sugars, which have their own effects on the response of in vitro cultures, (Chaplin 2008). Cooke (1977) mentioned that there was no significant difference in growth (fresh weight) of runner tips of Nephrolepis exaltata var. bostoniensis cultured on a modified Murashige and Skoog medium solidified by either agar or a new starch co-polymer agar substitute. The agar substitute promoted more shoot initiation than agar solidified medium. Although starch is the cheapest of the gelling agents, it is not expected to find universal acceptance as an alternative gelling agent because of its inferior gelling quality, lower clarity than agar and its metabolizable nature upon autoclaving, as starch yields sugars, which have their own osmotic or metabolic effects on cultures, which leads to softening of the media during the culture period (Babbar and Jain, 1998). Moreover, it softens even if the media are left un-inoculated (Tremblay and Tremblay, 1991). 

      Kodym and Zapata-Arias (2001) mentioned that cost reduction in tissue culture of banana 'Grande Naine' by shoot tip culture was achieved by replacing Gelrite in the medium with a starch/Gelrite mixture. Starches of corn or potato could partially substitute for Gelrite and agar.

      Naik and Sarkar (2001) reported that sago, a processed (gelatinized) edible starch, was successfully used as a gelling agent in culture medium of ten potato (Solanum tuberosum L.) genotypes. Sago starch provided a firm gelling surface throughout the entire culture period, and fostered optimum plantlet growth in terms of shoot height, number of nodes/plant, number of leaves and fresh mass. They showed that sago starch could be used as a substitute to agar in culture medium to substantially reduce the medium cost.

13 – Xanthan gum: Jain and Babbar (2006) stated that xanthan gum, a microbial desiccation-resistant polysaccharide prepared commercially by aerobic submerged fermentation from Xanthomonas campestris, has been successfully used as a solidifying agent for plant tissue culture media. Its suitability as a substitute to agar was demonstrated for in vitro seed germination, caulogenesis and rhizogenesis of Albizzia lebbeck, androgenesis in anther cultures of Datura innoxia, and somatic embryogenesis in callus cultures of Calliandra tweedii, Mimosaceae. Culture media used for eliciting these morphogenic responses were gelled with either 1% xanthan gum or 0.9% agar. Xanthan gum, like agar, supported all these responses. Xanthan gum being 3.6 and 13.5 times cheaper than the agar used in the present study and Difco-bacto agar respectively, provides a cost-effective alternative to agar. Media gelled with Xanthan gum (compared to agar-gelled medium) dry relatively at a slower rate than agar media. Therefore, these are better suited for long-term maintenance of cultures.

     This study aims to prove that it is possible to use cheaper substitutes instead of the expensive agar in the micropropagation by tissue culture techniques of two plants belonging to the Araceae family.

MATERIALS AND METHODS

      This work was carried out in the Tissue Culture Laboratory, Horticulture Research Institute, Agricultural Research Center, Giza, Egypt during the period from 2006 - 2007

       In vitro-produced explants of two aroids, Spathiphyllum wallisii and Anthurium scherzerianum were used as starting material s. Glass jars of 11.5 cm height x6.5 cm diameter with their polypropylene caps were used.

Table (a): Size (ml³) and weights of a substrate/jar (g).

     For each plant, 50 ml of the liquid Murashige and Skoog (1962) (MS) medium were poured in eight groups of these jars. Certain amounts of cotton, perlite, shredded straws of bagasse, rice and wheat, rice hull, bran and washed sand, as substrates, were put in each of these groups as shown in Table (a). Medium in a ninth group of jars was solidified with agar at 6 g/l. All media were supplemented with benzyl adenine (BA) at 3 ppm and kinetin (Kin) at 1 ppm.

     These 9 groups of jars represented treatments that were arranged in a completely randomized design. Each treatment comprised 12 replicates (jars).

Explants were inoculated on the media under aseptic conditions using a laminar airflow cabinet. Jars were incubated for four weeks at 25/20°C (day/night) ±2°C, 70% relative humidity. Two fluorescent tubes/shelf were installed at 30 cm above explants to provide light intensity of 2200-2400 lux at explant level for 16 h. daily.

      Data obtained for the two plants in the multiplication stage were: total number of shoots, shoot length (cm), total number of leaves, diameter of vegetative growth (cm) and total fresh weight (g). Shoot content of the photosynthetic pigments, i.e. chlorophyll a, chlorophyll b, total chlorophyll and carotenoids, were determined (in mg/g fresh weight) in agar, cotton and perlite in case of Spathiphyllum, and in the same media in addition to bagasse in case of Anthurium, according to Moran (1982). In the remainder media, these characters were undetectable.

       Data were statistically analyzed using SAS 1995 computer program, and means were compared by L. S. D. method according to Snedecor and Cochran (1980).

       Analysis of some characteristics of these substrates was conducted in the labs of the Horticultural Institute. One gram of each substrate was put in a beaker containing 200 cm3 of distilled water for 2 hours before these parameters were estimated. Results of this analysis are shown in Table (b).

Table (b): Analysis of the used substrates

RESULTS

Results are shown in Tables 1 and 2.

1 - Total number of shoots:

a - Spathiphyllum

The effect of media on total number of shoots was significant. The highest significant number in this regard (285.33 shoots) belonged to explants grown on the agar medium, followed with a significant difference with those grown on cotton (88.33 shoots) and perlite (44.00 shoots). Other media were significantly similar in their effect and scored the lowest values significantly.

b - Anthurium

The highest significant total number of shoots (94.33 shoots) was observed on the perlite medium. Both agar and cotton media were significantly similar and lower in their effect on the same character as they resulted in 32.00 and 25.67 shoots, respectively. Shoots produced on other media were significantly the least in number.

2 - Shoot length (cm):

a - Spathiphyllum

    The effect of media was significant on shoot length of Spathiphyllum. Longest shoots were those grown on rice straw (3.13 cm), followed without significant difference by those grown on agar, cotton and wheat straw (3.10, 2.50 and 2.37 cm, respectively). The second category, in a descending order, includes shoots grown on sand and bran, the lengths of which were 2.00 and 1.90 cm, respectively. The shortest shoots were obtained on rice hull (1.13 cm), proceeded by those grown on bagasse and perlite (1.60 and 1.70 cm, respectively).

b - Anthurium

    A significant influence was exerted by the media on shoot length of Anthurium. The longest shoots (2.60 cm) were observed on perlite, followed without significant difference by those grown on cotton (2.53 cm), agar (1.67 cm) and rice straw (1.63 cm). Rice hull medium produced the shortest shoots (0.60 cm) preceded without significant difference by those grown on bran (0.93 cm), sand (1.03 cm) wheat straw (1.13 cm) and bagasse (1.57 cm).  

  Table 1: Effect of medium substrate on Spathiphyllum wallisii shoots grown in vitro.

     Dia. of veg. = Diameter of vegetative growth

3 - Total number of leaves:

a - Spathiphyllum

     The media used in this study affected total number of leaves significantly. The significant highest number belonged to shoots grown on the agar medium (808.67leaves), followed with a significant difference with those grown on cotton (234.00 leaves). The total number of leaves produced on perlite (114.67 leaves) did not differ significantly either from the former record or from the corresponding values of the remainder media, the results of which were significantly the lowest.

Table 2: Effect of medium substrate on Anthurium scherzerianum shoots grown in vitro.

  Dia. of veg. = Diameter of vegetative growth

b - Anthurium

The effect of media on total number of leaves was significant. The highest significant number in this regard (266.67 leaves) belonged to shoots grown on perlite. The second value in a descending order was that of shoots grown on cotton (108.00 leaves), followed without any significant difference by those grown on agar (104.33 leaves). The lowest value was observed on shoots grown on rice hull (4.33 leaves). Other media resulted in number of leaves that did not differ significantly from the lowest value.   

4 - Diameter of vegetative growth (cm):

a - Spathiphyllum

Vegetative growth of shoots grown on agar medium occupied significantly the widest area with a diameter of 7.00 cm. Cotton and perlite media gave rise to the second and third widest diameters of vegetative growth in a significantly descending order (3.33 and 2.30 cm, respectively).

b - Anthurium

The effect of media on the diameter of vegetative growth was significant. The highest significant diameter (2.83 cm) belonged to shoots grown on perlite. The second record in a descending order was that of shoots grown on cotton (1.77 cm), followed without any significant difference by those grown on agar (1.60 cm). The lowest value was noticed on shoots grown on rice hull (0.08 cm). Other media resulted in vegetative growth that did not differ significantly in diameter from the lowest value.  

5 – Total fresh weight (g):

a - Spathiphyllum

Total fresh weight of vegetative growth (19.46 g) was significantly influenced by the media used. The heaviest fresh weight in this concern was a result of using agar as a medium. Shoots grown on all other media were significantly lighter than the former ones, though no significant difference was detected among them.

b - Anthurium

Media used in this study exerted a significant influence on total fresh weight of vegetative growth. The heaviest fresh weight was that of shoots grown on perlite (2.81 g). Fresh weight of shoots grown on agar (2.14 g) did not differ significantly from the corresponding weight of the former ones. Cotton medium resulted in shoots that weigh 1.34 g, insignificantly different from those grown on agar, but significantly lower than those grown on perlite. The lightest fresh weight (0.02 g) was that of shoots grown on rice hull. Fresh weight of shoots grown on the remainder media did not differ significantly from the former one.   

6 – Chemical determinations:

a – Spathiphyllum (Table 4):

    Parameters of the photosynthetic pigments, i.e. chlorophyll a, chlorophyll b, total chlorophyll and carotenoids, were determined in agar, cotton and perlite. In the remainder media these characters were undetectable. However, the estimated values were not influenced significantly by the growing media used in this study.

b – Anthurium (Table 5):

   Parameters of the photosynthetic pigments, i.e. chlorophyll a, chlorophyll b, total chlorophyll and carotenoids, were determined in agar, cotton, perlite and bagasse. In the remainder media these characters were undetectable. The

Table 4: Effect of medium substrate on photosynthetic pigments in Spathiphyllum wallisii shoots grown in vitro (mg/g fresh weight)

- = Undetectable amounts

Table 5: Effect of medium substrate on photosynthetic pigments in Anthurium scherzerianum shoots grown in vitro (mg/g fresh weight)

- = Undetectable amounts

highest significant values of these characters were observed in shoots grown on bagasse, followed without any significant difference by those grown on cotton. Corresponding values of shoots grown on perlite were not significantly different from those grown on agar. However, values of the two later media were significantly lower than those of the two former ones.

      DISCUSSION

      The highest records of total number of shoots, shoot length, total number of leaves, diameter of vegetative growth and total fresh weight of Spathiphyllum were observed in shoots grown on agar medium. Results of Anthurium shoots grown on agar medium did not differ significantly from the highest levels of shoot length and total fresh weight. Agar also achieved the second rank in total number of shoots, total number of leaves and the diameter of vegetative growth of Anthurium shoots.

       Length of Spathiphyllum and Anthurium shoots grown on cotton medium was not significantly different from the highest level. Cotton also achieved the second rank in total number of shoots, total number of leaves, diameter of vegetative growth and total fresh weight of Spathiphyllum and Anthurium shoots.

       In case of Spathiphyllum, perlite medium achieved the second rank in total fresh weight, and was not significantly lower than this category in total number of leaves. Records of Spathiphyllum shoots grown on perlite were lowered to the third rank in total number of shoots, shoot length and diameter of vegetative growth. On the other hand, the highest significant levels in total number of shoots, shoot length, total number of leaves, diameter of vegetative growth and total fresh weight of Anthurium were observed in shoots grown on perlite. 

      Values of the total fresh weight of Spathiphyllum shoos grown on bagasse medium significantly occupied the second rank in a descending order compared to the corresponding character in other media. The bagasse medium reached the third category in shoot length and total number of leaves, and the fourth one in total number of shoots and diameter of vegetative growth of Spathiphyllum. In case of Anthurium, values of all characters measured of shoots grown in the bagasse medium significantly reached the third rank in a descending order.

     Spathiphyllum shoots grown on straws of rice or wheat were significantly the longest, while those grown on bran or sand reached the second category in shoot length. Shoots grown in rice hull were significantly the shortest. Total fresh weight of Spathiphyllum shoots grown on rice straw, wheat straw, rice hull, bran or sand media occupied the second rank in a descending order. Total number of leaves of Spathiphyllum shoots grown in the same media was the third in a descending order. Total number of Spathiphyllum shoots and diameter of vegetative growth in the abovementioned media were significantly the least. Anthurium shoots grown on rice straw were significantly the longest, while those grown in wheat straw, bran or sand occupied the third rank in a descending order. Rice hull medium significantly produced the shortest Anthurium shoots. Other characters under investigation reached the third rank in rice straw, wheat straw, rice hull, bran or sand media.  

Parameters of the photosynthetic pigments, i.e. chlorophyll a, chlorophyll b, total chlorophyll and carotenoids, were not influenced significantly by media substrate in case of Spathiphyllum. On the contrary, the highest significant values of these characters were observed in Anthurium shoots grown on bagasse medium, followed without any significant difference by those grown on the cotton one. Corresponding values of Anthurium shoots grown on perlite were not significantly different from those grown on agar medium. However, values of the two later media were significantly lower than those of the two former ones.

     Among the many problems facing the tissue culture technique is the big sum of foreign currency needed to purchase the expensive agar from abroad. Workers over a long period of time have tried a lot of cheaper substitutes for agar especially when some problems of depending on agar were noticed. Results mentioned above are in accordance with the findings of those workers.

     Barbas et al (1993) reported that agar inhibited growth, induced mature leaf formation and necroses of the in vitro cultured shoots of hybrid walnut (Juglans regia X Juglans nigra). Agar altered the chemical composition of the medium as well as that of the explants. A pronounced accumulation of Na and several microelements was observed in leaves after 16 days of culture on agar, probably due to a disturbance in the K selectivity mechanism and membrane permeability. Mineral element accumulation was evident after growth inhibition. Lack of growth, mature foliar morphology, Na and microelement accumulation support the hypothesis that agar accelerates the ageing of in vitro propagated walnut trees. Nairn et al (1995) stated that tissue cultured Pinus radiata grown on a medium containing agar as the gelling agent display toxic symptoms and poor long-term shoot survival. A constituent could be separated from low molecular weight components of the agar responsible for the toxic symptoms by dialysis. It was identified as being an agaroid-type xylogalactan bearing pyruvate and sulfate substituents. Zimmerman et al. (1995) stated that agar, a seaweed derivative, is not suitable for culturing all plants.

     Moraes-Cerdeira et al. (1995) claimed that the cost of agar has prompted the search for an alternativemore economical medium support. Cotton fibers were tested as a substitute for agar in tissueculture. Effectiveness as a medium supportwas evaluated in terms of callus maintenance and shoot organogenesisusing Artemisia, Agrostis, and Taxus. Taxus and Agrostis callicultivated on liquid media with cotton fiber as medium support(25 ml of medium/g of cotton) grew better than callion agar (0.8% w/v). There were no significant differences inshoot organogenesis of Artemisia and Agrostis grown in medium supported by cotton from those grown in agar medium.

    Yi et al. (1998) described the use of perlite as an in vitro culture support in a Hyacinthus orientalis micropropagation system. Dissected bulb scale segments were cultured in Heller agar medium. After 16 weeks, regenerated bulblets over 5mm in diameter were subcultured on agar-solidified Heller medium or to the same liquid Heller medium plus perlite. They found that, in general, bulblet growth was better on the Heller liquid medium plus perlite than on the agar-solidified Heller medium, but the response varied with the cultivar. The use of perlite at the subculture stage also accelerated ex vitro growth of regenerated plantlets transplanted into a soil mixture.

   On the contrary, agar was praised by some authors such as Punia et al (2000) when they studied the effects of using liquid media with support matrices (nylon cloth, muslin cloth, glass wool or filter paper) on sugarcane (cultivars CoH92 and CoH99) shoot multiplication. They found that shoot multiplication decreased sharply when liquid media containing nylon and muslin cloth, glasswool, or filter paper were used.

   Gebre and Sathyanarayana (2001) conducted a study to compare the efficacy of cheap alternative gelling agents, tapioca (from tubers of Manihot esculenta) and sago (from the stem pith of Metroxylon sagu), and agar. They reported that the type of gelling agent significantly affected in vitro plant regeneration of nodal explants of potato. Shoot height in agar reached 8.9 cm with 10.5 nodes, while it ranged between 8.9 and 9.8 cm in tapioca with 11.3-12.1 nodes. Sago-based medium was generally unfavourable for in vitro shoot proliferation. The effect of gelling agent was more pronounced on microtuber fresh weight than on microtuber number. A differential increase in weight occurred in the media solidified with either tapioca or sago. Within a concentration of 11-14% of tapioca, the existence of a favourable osmotic environment and better carbohydrate and ionic supplement is suspected, which may have improved cell growth and morphogenesis. The results showed the possibility of a successful use of tapioca for in vitro culture of potato.

    El-Kazzaz and El-Bahr (2003) found that the best medium for developing the date palm cv. Samany plantlets in vitro was a liquid half MS medium with perlite as substrate, which gave the highest shoot length.

     Saadawy (2005) ascertained that in search of cheaper substrates instead of agar, perlite gave the significant heaviest Gerbera shoots, the highest number of shoots and the greatest significant leaf number. Gerbera shoots grown on perlite had the highest content of total chlorophyll. In case of Lilium, the significant heaviest shoots and the highest number of shoots were a result of using cotton as a substrate. Longest Lilium shoots were those grown on peat moss. Greatest significant leaf number and the highest content of total chlorophyll were detected in shoots grown on cotton.

Conclusion

The use of cheaper substrates such as cotton, perlite or bagasse could be recommended as a replacement for agar, especially in commercial tissue culture labs for of the micropropagation of the two aroids Spathiphyllum wallisii and Anthurium scherzerianum.

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ملائمة بعض بدائل الآجار لإکثار الأنتوریوم والاسباثیفیللم بتقنیة زراعة الأنسجة فی مرحلة الإکثار

فیصل محمد سعداوى*- منى أحمد درویش**-  هند مصطفى فهمى** -               هبة حسن محمد حسن*

* قسم بحوث نباتات الزینة-  معهد بحوث البساتین- مرکز البحوث الزراعیة الجیزة- مصر.

**  قسم الزینة-  کلیة الزراعة- جامعة القاهرة-  الجیزة-  مصر.

أجریت هذه الدراسة فی محاولة للبحث عن بدیل أرخص للآجار. وقد اختبرت الکثیر من المواد لتکون مهدا للزراعة مع محلول موراشیج وسکوج وقورنت ببیئة موراشیج وسکوج المضاف إلیها الآجار. زرعت منفصلات نباتیة لإثنین من نباتات العائلة القلقاسیة هما سباثیفیللم وأنتوریوم على هذه البیئات فی ظروف معقمة.

تسببت البیئة المضاف لها الآجار فی إنتاج أکبر عدد من الأفرع ذات الطول الأکبر والعدد الأکبر من الأوراق والقطر الأکبر للنمو الخضری وأکبر وزن کلى رطب للنمو الخضری لنبات سباثیفیللم. ومن الناحیة المعنویة لم تختلف نتائج أفرع الأنتوریوم النامیة على البیئة المضاف لها الآجار عن المستوى الأعلى من حیث طول الأفرع والوزن الرطب للنمو الخضری. کما أحرزت البیئة المضاف لها الآجار المرتبة الثانیة بالنسبة للعدد الکلى للأفرع وللأوراق والقطر الأکبر للنمو الخضری لنبات الأنتوریوم.

لم یختلف طول أفرع السباثیفیللم والأنتوریوم النامیة على بیئة موراشیج وسکوج المضافة على القطن بدرجة معنویة عن المستوى الأعلى للطول. کما أحرزت بیئة القطن المرتبة الثانیة بالنسبة للعدد الکلى للأفرع والأوراق وقطر النمو الخضری والوزن الکلى الرطب لکلا النباتین.

أحرزت بیئة موراشیج وسکوج المضافة على البیرلایت المرتبة الثانیة من حیث الوزن الکلى الرطب، ولم تقل من الناحیة المعنویة عن هذه المرتبة بالنسبة للعدد الکلى للأوراق لنبات سباثیفیللم. وقد انخفضت نتائج أفرع سباثیفیللم النامیة على البیرلایت إلى المرتبة الثالثة من حیث العدد الکلى للأفرع، وطول الأفرع، وقطر النمو الخضری. ومن ناحیة أخرى، فقد لوحظت المستویات الأعلى من الناحیة المعنویة من حیث العدد الکلى للأفرع، وطول الفرع، والعدد الکلى للأوراق، وقطر النمو الخضری، والوزن الکلى الرطب لنبات الأنتوریوم فی الأفرع النامیة على البیرلایت.

احتلت قیمة الوزن الکلى الرطب لنبات سباثیفیللم النامیة على بیئة موراشیج وسکوج المضافة على مصاصة القصب المقطعة المرتبة الثانیة بدرجة معنویة مقارنة بالقیم المناظرة فی البیئات الأخرى بترتیب تنازلی. کما أحرزت بیئة مصاصة القصب المرتبة الثالثة من حیث طول الفرع، والعدد الکلى للأوراق، والمرتبة الرابعة بالنسبة للعدد الکلى للأفرع، وقطر النمو الخضری فی نبات سباثیفیللم. وفى حالة نبات الأنتوریوم فقد احتلت قیم کل الصفات المقاسة للأفرع النامیة على مصاصة القصب المرتبة الثالثة معنویا بالترتیب التنازلی.

کانت أفرع السباثیفیللم النامیة على بیئة موراشیج وسکوج المضافة على قش الأرز أو قش القمح هی الأطول بدرجة معنویة بینما کانت أفرع الأنتوریوم النامیة على البیئة المحتویة على قش الأرز هی الأطول معنویا.

لم تتأثر قیم صبغات التمثیل الضوئی ( کلوروفیل "أ" وکلوروفیل "ب" والکلوروفیل الکلى والکاروتینویدات) بدرجة معنویة بالمهد المستعمل فی البیئة فی حالة نبات سباثیفیللم. وعلى العکس، فقد لوحظت أعلى هذه القیم فی أفرع الأنتوریوم النامیة على البیئة المحتویة على مصاصة القصب، یلیها بدون فرق معنوی تلک النامیة البیئة المحتویة على القطن. ولم تختلف القیم المذکورة لأفرع الأنتوریوم النامیة على البیئة المحتویة على البیرلایت بدرجة معنویة عن تلک النامیة البیئة المحتویة على الآجار. یلاحظ أن قیم الصفات المذکورة على البیئتین الأخیرتین کانت أقل معنویا من نظیرتها على البیئتین السابقتین. 

التوصیة: یمکن النصح باستعمال مهاد أرخص للبیئة مثل القطن والبیرلایت ومصاصة القصب بدیلا للآجار، خاصة فی معامل زراعة الأنسجة التجاریة.