The effect of IAA and other auxins on the growth of date palm(Phoenix dactylifera L.) shoot tips and callus

Abstract

Shoot tips and callus of some cultivars of date palms (Phoenix dactylifera L.) were cultured on MS (Murashige and Skoog) and GM5 (Gambourge B5) media by using auxin IAA mainly and other auxins NAA, 2,4-D and IBA. The results showed a significant superiority of cultivar Hillawi than CVS. Zahdi, Barhee, Braim and Khasab for growth value and dry weight of shoot tip segments. There is a significant superiority of auxin IAA in MS medium than both IAA and 2,4-D in GM5 for growth value and dry weight for shoot tip segments of CV. Hillawi, and significant superiority of auxin IAA at level 100mg/L than auxin NAA (100mg/L) for growth value and dry weight of shoot tip segments of cv. Hillawi , and also a significant superiority of auxin IAA (10mg/L) than IAA at levels (0, 25, 100mg/L) for growth value an dry weight of both shoot tips segments and leaves primordia of cv. Sayer. Finally there is a significant superiority of auxin IAA (10mg/L) than auxins NAA, IBA and 2,4-D at the same levels for growth value of embryogenic callus of cvs. Barhee and Hillawi whereas the superiority was for both IAA, NAA, than the rest auxins for embryo length for cvs. Barhee and Hillawi. ٢٠٠٧ ةنسلا ١ :ددعلا ٦:دلا رمتلا ةلنخ ثابحلأ ةرصبلا ةلمج 2 Introduction Date palm phoenix dactylifera L. is the important tree of which man has been interested since the ancient ages especially in Iraq; the success of its agriculture is due to its tolerance to high temperature and salinity [Saker et al., 1997]. The continuous decreasing in numbers of date palm trees in Basrah by many reasons enhancing to find an other way for propagation of date palm, this way is tissue culture technique. The first atempt was begun by Shroeder (1970) who used shoot tips and axillary buds of date palm as explants. Tisserat (1979, 1981) obtained date palm plants by somatic embryogenesis from the culturing of shoot tips and auxillary buds on MS (Murashige and Skoog) medium supplemented with 100mg/L (2,4D) . Sharma et al., (1984, 1986) also obtained callus from shoot tips and axillary buds on MS medium involving the change of NaH2PO4. 2H2O from 170mg to 200mg/L. Mater (1986) obtained a yellowish nodular callus after 6-8 months of subculturing of initial callus from shoot tips of cultivars: Hillawi and Barhee on MS medium supplemented with BA (Benzyl adenine) and Kinetin (2mg/L each) , 2,4-D (100mg/L) to initiate callus and then NAA (Naphthalene acetic acid) 0.1mg/L instead of 2,4-D to form somatic embryos. Abo El-Nil (1986) reported the initiation of callus from shoot tips and axillary buds on MS medium supplemented with 2,ip (2, Isopentenyl adenine) and NAA. Al'utbi (1998) obtained callus for many local cultivars from shoot tips and axillary buds on MS medium supplemented with 2,4-D (100mg/L), Kinetin, Benzyl adenine (2mg/L for each) then substitution of 2,4-D by low levels of NAA enhancing the formation of somatic embryos then plantlets. Omar (1988a) observed white grainy callus initiation from leaf segments in nutrient medium enriched with 3mg/2ip and 100mg/NAA. The aim of this study is to explore the effect of IAA (Indole acetic acid) as natural auxin on shoot tip and callus of date palm In Vitro in addition to ( 2,4D) and (NAA) those were believed to have blemish of carcinogenic for the first and weakness for the latter. ٢٠٠٧ ةنسلا ١ :ددعلا ٦:دلا رمتلا ةلنخ ثابحلأ ةرصبلا ةلمج 3 Preparation of explants:Date palm offshoots (2-3 years aged) cvs. Hillawi, Sayer, Zahdi, Khasab, Braim and Barhee were brought from Abu-Alkhaseeb orchards and dissected acropetally, shoot tips were excised and trimmed to shoot tip disc (8 x 3mm) preserved in antioxidant solution (150mg/L citric acid + 100mg/L Ascorbic acid) , then sterilized in infestant solution of sodium hypochlorite NaOCl 20% with two drops of tween-20 for 20 minutes washing with sterilized distilled water three times and culturing on fresh medium; all operations were performed in laminar air flow. Preparation of medium : A) MS medium according to (Murashige and Skoog, 1962). B) Gambourge B5 medium (Gambourge and Eveligh, 1968). Both types of media were supplemented with the following materials (mg/L), Sucrose (30000), NaH2PO4 (170), Meso-Inositol (100), thiamine-HCl (0.5), activated charcoal (3000), Agar (8000), Kinetin (3), Benzyl adenine (BA) (1): Auxins were added according to statistical experiments requirements as follows:1) Auxins IAA (100mg/L) was added to MS medium for the growth of shoot tips of different varieties of date palm. 2) IAA, 2,4-D (100mg/L) were added to MS and GM5 media for the growth of shoot tips of cv. Hillawi. [MS medium + auxin IAA], [MS medium + auxin 2,4-D], [Gambourge B5 medium + auxin IAA], [Gambourge B5 medium + auxin 2,4-D]. 3) Auxins, NAA , IAA (100mg/L for each) were added to MS and GM5 media for the growth of shoot tips cv. Hillawi in combinations: [MS medium + Auxin NAA] , [MS medium + auxin IAA] [Gambourge B5 + Auxin NAA], [Gambourge B5 + Auxin IAA] . ٢٠٠٧ ةنسلا ١ :ددعلا ٦:دلا رمتلا ةلنخ ثابحلأ ةرصبلا ةلمج 4 4) IAA was added at level (0, 25, 50, 100) mg to MS medium for the growth of shoot tips and leaf primordia of cv. Sayer. 5) Auxins IAA, IBA, NAA, 2,4-D were added to MS medium for the growth of embryogenic callus and somatic embryos cvs. Barhee and Hillawi. pH was adjusted at 5.7, Agar was added. After heating to 95°C the solution was cooled and dispensed in containers (test tubes and conical flasks). These containers were closed with cotton and aluminum foil , then they were autoclaved at 121°C and 1.05kg/cm for 20 minutes. C) Subculture of callus : Callus was subcultured at periods of 60 days continuously on MS medium supplemented with 2,4-D or NAA (100mg/L) in addition to benzyl adenin (1mg/L) and kinetin (3mg/L). d) All contents of MS medium are used for the preparation of a medium for somatic embryos formation; except auxins IAA, 2,4-D, NAA were removed with their concentration (100mg/L) and only NAA (1mg/L), kinetin (3mg/L) and BA (1mg/L) were added instead of them. Growth value was calculated by the following formula: Final fresh weight Growth value =----------------------------------Initial fresh weight (Almehdi, 1976) All experiments were performed according to C.R.D. (Complete Randomized Design) (5-10 replicates) and the results were analyzed according RLSD. (AL-Rawi and Khalafullah, 1980). ٢٠٠٧ ةنسلا ١ :ددعلا ٦:دلا رمتلا ةلنخ ثابحلأ ةرصبلا ةلمج 5 Results 1Effect of the addition of auxin IAA on the growth of shoot tips cvs. Hillawi, Braim, Khasab, Barhee and Zahdi to compare their responses. The results showed (Table 1) that significant superiority of cv. Hillawi in growth value (5.18) than all other treatments: Zahdi, Barhee, Braim, Khasab (2.75, 2.13, 1.87, 1.40), respectively, but there is no significant differences among the rest cultivars, also there is a significant superiority of cv. Hillawi in dry weight (62mg) than all other treatments: Barhee, Braim, Khasab and Zahdi (29mg, 17.40mg, 12.40mg and 35.40mg), respectively, and there is no significant difference among the rest treatments. Table 1: Effect of response of five cultivars of date palm for the growth of shoot tips segments (300 gm as mean) on MS medium containing 100mg/L auxin IAA ( after two months-culture period) Treatments (five cultivars of date palm) Measurement Hillawi Braim Khasab Barhee Zahdi Growth value 5.18 *a 1.87 b 1.40 b 2.13 b 2.75 b Dry weight (mg) 62.0 a 17.40 b 12.40 b 29.00 b 35.40 b *Values with the same letter are not significantly different by RLSD analysis (p ≤ 0.05). 2Effect of the addition of auxins IAA to MS and GM5 media on the growth of shoot tip cv. Hillawi. The results showed (Fig. 1) a significant superiority at p ≤ 0.05 of treatment [IAA in MS medium] in growth value (22.36) than the treatment [IAA in GM5 medium] (6.17), there is also a significant superiority at p≤0.05 of treatment [IAA in MS medium] in dry weight (144.80 mg) than the treatment [IAA in GM5 medium] 57.80mg. ٢٠٠٧ ةنسلا ١ :ددعلا ٦:دلا رمتلا ةلنخ ثابحلأ ةرصبلا ةلمج 6 Fig. 1: Effect of the addition of auxin IAA to MS and GM5 media on the growth of shoot tip segments (300mg as mean) cv. Hillawi (after two months-culture period) Agrowth value Bdry weight The similar letters indicate that there is no significant difference by RLSD analysis (p ≤0.05). 3Effect of the addition of auxin IAA, NAA to MS medium on the growth of shoot tip segments cv. Hillawi. Results showed a significant superiority of treatment [IAA, 100mg/L] than treatment [NAA, 100mg/L] for growth value and dry weight (table 2). Table 2: Effect of the addition of IAA , NAA to MS medium on the shoot tip segments ( 300mg as mean ) cv. Hillawi (after two months-culture period) Treatment 100mg/L Measurements IAA NAA Growth value 21.99 a 5.00 *b Dry weight (mg) 171.30 a 45.55 b *Values with the same letter are not significantly different by RLSD analysis (p ≤ 0.05). 4Effect of the addition of different levels of auxin IAA on both shoot tip segments and leaves primordia of cv. Sayer. 0 20 40 60 80 100 120 140 160 a

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