GROWTH OF DATE PALM CALLUS AS AFFECTED BY AMINOACIDS AS ORGANIC NITROGEN SOURCE

Abdel-Rahim, E.A.I; Abdel-Fatah, O.M.I; EI-Shemy, H.A.I and Abd EI-Samei, M.B2

1- Agric. Biochem. Dept., Fac. Agric. Cairo Univ., Giza, Egypt.

2- Agric. Chem. Dept., Fac. Agric. Minia Univ., EI-Minia, Egypt.

ABSTRACT

MS-medium that contained kinetin 0.5 mgIL with 2 mgIL 2,4-D wasthe best combination of growth regulators in callus production of datepalms. The effect of methionine, tryptophane, alanine, glutamic acid andcasein hydrolysate on growth and production of callus were tested. Allamino acids treatments steadily decreased both fresh and dry weights ofcalli compared with control. Also, it was noticed that methionine andtryptophane treatment gave the lowest values of both fresh and dry weights.However, alanine and glutamic acid added to the nutrient medium decreasedthe growth of callus compared with other amino acids. Fresh and dryweights of callus derived from leaf primordia of terminal buds stem tissuewere determined as affected by casein hydrolysate which supplemented toculture medium. Casein hydrolysate concentrations were added to themedium at 0.5, 1.0, 1.5 or 2.0 gIL. The obtained results revealed that, withall treatments, the fresh and dry weights were gradually increased with~asein hydrolysate concentration increased and reached their maximum with2.0 gIL casein hydrolysate.

"Additional Index Words": Tissue culture, Phoenix dactylifera L.,casein hydrolysate, glutamic acid, phenylalanine, tryptophane, ornithine,methionine, arginine, alanine.

INTRODUCTION

Cultured cells are normally capable of synthesizing all of requiredamino acids yet, the addition of amino acid or amino acids mixture may beused to stimulate cell growth and facilitate plant regeneration (Mohamed,1996). L-glutamine can serve as the sole source of nitrogen which can betaken more rapidly than inorganic nitrogen (Thorn et aI., 1980). Hussein etal. (1994) studied the effect of some amino acids on the growth of Datura

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stramoinium cultured on MS-medium with 1 mg/L of both 2,4-D and kinetinand subcultured on fresh medium containing some amino acids. They foundthat, the fresh and dry weight of calli was reduced. Kamada and Harada(1979) reported that, at the addition of individual amino acids to carrotcallus culture, alanine stimulated somatic embryogensis followed byglutamine, asparagine, aspartic acid, arginine and proline. Abou El-Nil(1989) indicated that amino acids stimulated callus growth and ranked, asfollows: glutamine, asparagine, arginine, serine, glycin and alanine.Glutamine caused doubling callus growth compared to control.

The effect of casein hydrolysate (amino acids mixture) as a solenitrogen sources on callus growth was reported by Heimer and Filner (1970)on tobacco callus. Zenk et aI., (1975) found that, when cultured medium wassupplemented with casein hydrolysate at level greater that 4 g/L, callusgrowth was stimulated in Morinda citrifolia. Casein hydrolysate was used asa sole nitrogen source for beans (Crocomo et aI., 1976), carrot (Wetherelland Dougall, 1976) and fenugreek (Singh et aI., 1981). Cardi and Monti(1993) found that, the addition of casein hydrolysate at 2 g/L is importantfor callus production from pea. Also, in a study on kidney bean and peacallus, Saker (1995) reported that, the addition of 2 g/L casein hydrolysateto culture medium enhanced callus growth.

This study was carried out to determine the activity of some aminoacids and casein hydrolysate in date palm tissue culture. .

MATERIALS AND METHODS

Culture media:

The composition and concentration of the medium used in this workwere according to Murashige and Skoog (1962) Media were autoclaved at121°C and 1.2 Kg/cm2 for 20 min. The water used for preparation of themedia was double distilled and sterile. The medium was solidified with

0.7% agar, pH was adjusted to 5.8 before autoclaving. The prepared mediawere poured into heat sterilized 100 ml glass containers. Each containedabout 25 ml solidified medium.

Callus Initiation and Maintenance:

Callus cultures from date palm (Phoenix dactvlifera L.) from leafprimordia of terminal buds stem tissue was initiated on the MS-media.

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Certain amino acids included glutamic acid, phenylalanine, tryptophane,ornithine, methionine, arginine and alanine were used.

Subculturing was carried out after 4 weeks by cutting the callus intosmall pieces each about 200 mg and transferring them to fresh nutrient MS-basal medium supplemented with 2 mg/L 2,4-D, 0.5 mg/L kinetin and 10mM of each amino acid individually.

To study the effect of casein hydrolysate on date palm callus, callustissue was transferred to fresh MS-medium, supplemented with 2 mg/L 2,4-D, 0.5 mg/L kinetin and different concentrations of casein hydrolysate (0.5,1.0, 1.5 and 2 g/L). Cultures were incubated in a 16-hours photoperiod at24-26 QC.The growth was determined at the end of the 4 weeks incubationperiod (6 replicates).

The obtained data from the present study were subjected to statisticalanalysis performed using standard deviation (S.D) according to the methoddescribed by Snedecor and Cochran (1980).

RESULTS AND DISCUSSION

Effect of amino acids on Callus Growth:

The influence of amino acids supplemented to the culture medium ongrowth of initiated callus tissues of date palm were studied. Cflllistockgrown onto MS-medium contained 2 mg/L 2,4-D and 0.5 mg/L kinetinsupplemented with 10 mM/L of different amino acids. From Table (1), itcould be noticed that, the fresh weight and growth rate of callus tissues weredecreased as affected by amino acids supplemented to culture mediumcompared with control. Filner (1966) reported that the growth of tobaccocallus in culture was inhibited by a wide variety of amino acids added singlyto the nitrate medium on which the cells were grown, and that the inhibitioncould be attributed to repression of the nitrate reductase activity in the cells.Also, Fukunaga and King (1982) reported that, some single aminocompounds inhibited the growth of datura cells at points in metabolismother than nitrate assimilation and that in no case could growth and nitratereductase activity inhibition be causally unequivocally.

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Table (1): Effect of amino acids on the fresh weight (gm/inoculum) andgrowth rate (mg/day) of the callus tissue of date palm cultured 4 weeks.

Each value was the average of 6 replicates, % at control

Comparing the effect of amino acids supplementations on the growth,it could be noticed that tryptophane methionine and treatments gave thelowest values of. both fresh and growth rate. However, arginine, alanine,asparagine and glutamic acid gave the highest values of both fresh andgrowth rate. In accordance, Filner (1966) showed that, alanine, asparagine,glutamic acid and arginine did not inhibit the growth when they were addedto tobacco cells growing with nitrate as a sole nitrogen source. On the otherhand, Mohamed (1996) reported that, glutamic acid, methionine,tryptophane, phenylalanine and arginine inhibited the growth of callustissue cultures of fenugreek. In contrast, Abou El-Nil (1989) reported that,growth of date palm callus tissue was stimulated by adding of amino acidsspecifically glutamine. This stimulation suggested that organic nitrogen wasa growth-limiting factor in date palm cultures.

Effect of casein hydrolysate on Callus Growth:

The effects of different supplementations of casein hydrolysate on thegrowth of date palm callus was determined (Table 2). Fresh weight andgrowth rate of calli gave increasing values during cultivation period. It isfound that, when casein hydrolysate concentration increased in the medium,the growth of the tissue as fresh weight and growth rate were increased.

.Similarresults for the positiveeffectsof caseinhydrolysateon callusgrowthwere reported by Crocomo et aI., (1976), and Mok and Mok (1985) on

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Amino acids Callus fresh weight:i: % Callus growth rate %10 mM/L SD mg/day

gmlinoculumControl 2.630 :l: 0.110 100 87.667 100

Glutamic acid 1.818 :l: 0.071 69 60.600 69Methionine 1.757 :l: 0.072 66 58.567 66Tryptophane 1.403 :l:0.069 53 46.767 53

Phenylalanine 1.800 :l: 0.072 68 60.000 68Ornithine 2.104 :l: 0.094 80 70.133 80Arginine 2.103 :l: 0.090 80 60.267 69Alanine 2.041 :l: 0.089 78 68.033 78

kidney bean, Murashige and Skoog (1962) on tobacco and Singh et aI.,(1981) and Mohamed (1996) on fenugreek.

Table (2): Effect of different concentrations of casein hydrolysate on freshweight (gm/inoculum) and growth rate (mg/day) of the callus tissue of datepalm cultured 4 weeks.

Each value was the average of 6 replicates, % at control

The present data clearly indicated that, all concentrations of caseintreatment steadily increase both fresh weight and growth rate of callicompared with control. Two g/L casein hydrolysate treatment gave thehighest value of both fresh weight and growth rate. On the other hand,Matsumoto et aI., (1976) showed that, the requirement of amino acids forplant tissue culture could be estimated by adding different amounts ofprotein hydrolysate. The enhancement of growth or morphogenesis could beexplored further by using a mixture of amino acids. They provide plant cellswith an immediately available source of nitrogen which generally can betaken up by the cells more rapidly than inorganic nitrogen (Thorn et aI.,1980).

ACKNOWLEDGMENT:

The authors would like to thank Prof. Dr. Abel-Rehim F.A., Dept. ofBiochem., Fac. of Agric., Cairo Univ., Giza Egypt, for his help during thiswork.

LITERATURE CITED

Abou El-Nil M. 1989.The effects of amino acid nitrogen on growth ofdate palm callus. Second Symposium on date palm, K.F.U., AI-Hassa, SaudiArabia, 59-65.

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Hydrolysate Callusfreshweight:f: SD % Callus growth rate %Conc.gm!L gmfinoculum mg/day

Control 1.523 :f: 0.104 100 50.767 1000.5 1.618 :f: 0.110 106 53.933 1061.0 1.618 :f: 0.142 164 83.133 1641.5 2.625 :f: 0.137 172 87.500 1722.0 2.741 :f: 0.141 180 91.367 180

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Murashige T. and Skoog F. 1962.A revised medium for rapid growthand bioassays with tobacco tissue culture. PhysioI. Plant., 15:473.

Saker M.M. 1995. Studies on tissue cultures of some legumes. Ph.D.Thesis, Fac. Sci., Tanta Univ., Tanta, Egypt.

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Sendecor G.W. and Cochran W.G. 1980. Statistical Methods. 7th Ed.Iowa State Univ. Press, Iowa, USA.

Singh N.N., Kokate C.K. and Tipnis H:P. 1981.A note on developmentof callus cultures of Trigonellsfoenum-graecum for diosgenin biproduction.Indian Drugs., 19(1):25.

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