EXPLANT RESPONSES OF AGED TREES AND OFFSHOOTS OF DATE PALM IN MICROPROPAGATION.

EXPLANT RESPONSES OF AGED TREES AND OFFSHOOTS OF DATE PALM IN MICROPROPAGATION.

 

Hamdy Ahmed Emara

Plant Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), Minufiya University, Sadat City, Egypt.

 

           

ABSTRACT

Micropropagation of date palm (Phoenix dactylifera L. cv. Samany) through somatic embryogenesis and organogenesis methods have been studied in this investigation. Aged tree and juvenile offshoot of date palm were used as source of explants. Shoot tips and leaf primordia were isolated from those two sources to be used as two types of explants. The response of the two types of explants was examined when they cultured on two different nutrient media (M1 and M2), where M1 medium was modified MS contained NOA (5mg/l), NAA (5mg/l), Kin (3mg/l) and 2iP (3mg/l) and M2 medium was  MS contained 2,4-D (100 mg/l) and 2IP (3 mg/l).

Results indicated that there was no significant difference between shoot tips and leaf primordia derived from either aged tree or juvenile offshoot for their effect on survival percentage, while, the degree of oxidative browning on M1 medium was significantly lower than M2 medium. After 8 weeks or even 4 months, all examined explants formed callus on M2 medium as compared to M1 medium, which showed no callus formation. Interestingly, results proved that leaf primordia of both sources cultured on M1 medium stimulated direct organogenesis and somatic embryogenesis after 4 months of incubation. In that concern, shoot tips of both sources did not observe any direct morphogenetic responses. Growth and development of the previously obtained adventitious buds as well as repetitive somatic embryos have been optimized on hormone-free M1 medium. Roots were induced in MS basal medium contained NAA (0.2 m /L), and mostly, the addition of charcoal to the rooting medium had no visible effect on root formation. At acclimatization stage, it was found that, using of induced the maximum survival rate and growth vigor for plantlets after 3 months under greenhouse conditions compared to those produced under the other tested soil mixtures. Finally, results strongly reflected that, leaf primordia of aged tree or offshoots are a promising explants on M1 medium for the mass production of true-to- type plants since the callus stage is avoided.

          Key words: Phoenix dactylifera L., adult, in vitro, tissue culture, embryogenesis,   organogenesis.

  

           

INTRODUCTION

Date palm is monocotyledonous plant that matures into a single shoot. It propagates naturally by seed and offshoots. Usage of seed propagation is very limited since varieties do not breed true-to-type. The date palm is long lived and produces relatively few offshoots suitable for transplanting in its lifetime; consequently, offshoots of the best varieties are always in short supply and command high prices (Zaid and De Wet 2005). About two decades ago, scientists succeeded in propagating date palm through somatic embryogenesis via callus and axillary bud multiplication methods (Tisserat 1979, Drira, 1983 and Veramendi and Navarro, 1996). Somatic embryos could be formed indirectly through callus or directly without any intervening callus phase (Hegazy 2003). Direct somatic and repetitive embryogenesis showed the best balance of high propagation rates with relatively few off-types (Phillips and Hubstenberger 1995). In vitro propagation of the date palm is dependent on using the offshoots as a common source of the explant materials. On the other hand, little work has been made on using adult aged date palm trees as a source of explants. Availability of tissue culture material from adult plants at a reasonable cost will ease constraints on the replacement of old plantations, where tall palms have become costly to maintain. This approach will be beneficial in multiplying the good cultivars of date palm.

This study aimed to investigate in detail the in vitro responses of offshoots and aged trees (as two sources of explants) of date palm (Phoenix dactylifera L cv. Samany) using two types of explant (shoot tips and leaf primordia) from each source. The responses of these explants were examined in two different nutrient media.

 

MATERIALS AND METHODS

 

This work was carried out in the Plant Biotechnology Department of the Genetic Engineering and Biotechnology Research Institute (GEBRI), Sadat City, Minufiya University, during the period 2004- 2006.

         Explant materials used in this study were obtained from 4 years- old offshoots and about 60 years-old trees of aged female date palm cultivar Samany having high quality and grown at Giza governorate. The leaves and fiber sheath of both sources were removed acropetally with a hatchet and a sharp knife. During dissection process, 70% Ethyl alcohol was sprayed over cutter and plant material. when the final size were about3.0 to4.0 cm in width, and 8.0 to 10.0 cm in length, shoot tips were excised and soaked in sterilized antioxidant solution (150 mg/L each of citric acid and ascorbic acid).Explants were kept in the refrigerator at (5.0 C°) until the surface sterilization procedure is performed .Explants were surface sterilized by soaking for 25 minutes in 40% Clorox (2% Na OCL) containing two drops of tween-20 per 100 ml. Explants were then rinsed with sterile distilled water. Another post- treatment used was 0.1 % Mercuric chloride solution containing two drops of tween -20 per 100 ml for 5 minutes. Explants were then rinsed three times with sterile distilled water and finally soaked in sterilized anti-oxidant solution till dissecting. Additional leaves were removed when the cluster of very little leaves of the apex is reached, leaf primordial were isolated one by one obtaining the shoot tip with a base of meristele tissue, and then, shoot tips and leaf primordia were cultured individually with a good contact on the surface of the medium.

Nutrient Media

Shoot tip and leaf primordium explants isolated from offshoots and aged trees were cultured on two types of media:-

 

 M1 medium: MS medium was modified by Hegazy (2003) and contained; The MS basal medium (Murashige and Skooge, 1962)  supplemented with citric acid (75 mg/L), ascorbic acid (75 mg/L), PVP (1500 mg/L), Ca-pantothenate (2.5 mg/L), asparagen (100 mg/L), glutamine (200 mg/L), myo-inositol (125 mg/L), thiamine.HCl (0.5 mg/L), biotin (0.2 mg/L), Na H₂ PO₄ (170 mg/L), adenine sulfate, 2H20 (20 mg/L), naphthoxy acetic acid 5.0 mg/L (NOA), naphthalene acetic acid 5.O mg/L (NAA) isopentenyl adenine 3.O mg/L (2iP) kinetin 3.0 mg/L (kin), sucrose (40 g/L), activated charcoal (1.5 g/L) and phytagel (1.5 g/L).

 

M2 medium: MS medium was used by Tisserat (1979) and contained MS basal medium supplemented with 2,4-D (100 mg/L) and 2iP (3 mg/L).

The pH of all media were adjusted to 5.7 with 0.1 N KOH or HCI before the addition of phytagel and dispensed either into 25X20 mm culture tubes in aliquots of 20 ml per tube and capped with Belico Kaputs or into jars in aliquots of 50 ml per vessel. Jars were covered with polypropylene closure. Media were then autoclaved for 15 minutes for test tubes and 20 minutes for jars at 121C° and air pressure of 1.2 kg/cm2.

 

Growth parameters:

Data were taken after 8 weeks as percentage of survival and callus formation as well as degree of browning and growth value. After 4 months, percentage of callus formation, direct embryogenesis and direct adventitious buds were recorded.

Growth and development of adventitious buds as well as repetitive embryos were optimized on hormone-free M1 medium.

Root formation:

MS basal medium containing 0.2 mg/L NAA either with activated charcoal or free were tested for root formation. Percentage of root formation was recorded after 1, 2, and 3 months.

 

Culture conditions:

       Cultures were incubated in the growth room in total darkness at 25 ^oC ±1. These cultures were transferred to fresh medium after 2 weeks and then, recultured twice at 4- week's intervals even if no growth was visible. Cultures were transferred under conditions of light intensity 1500 Lux derived from ordinary fluorescent lamps for 16 hours photoperiod. Light intensity (3000 lux) was used in rooting stage.

Degree of browning: It was determined according to the rate of scalling by Pottino (1981), which included, no browning (1), average browning (2) and high browning (3).

Growth value: Explants growth value was determined according to the equation of Ziv (1992).

Growth vigor:  Estimated as scores according to the method described by Pottino (1981) as follows: 

1- Weak growth 2- Below average growth 3- Average growth 4- Above average growth 5- Excellent growth.

Acclimatization:

        Plantlets developed well root and shoot system were acclimatized in plastic pots contained different soil mixtures:  peat moss, peat moss and sand (1: 1, v/v), peat moss and vermiculite (1: 1, v/v), peat moss and perlite (1: 1, v/v) or clay. Survival percentage of plantlets was recorded after 3 months.

Statistical analysis:

Data were statistically analyzed by two factorial randomized complete block design using the SAS (1988) package. The Least Significant Differences among levels of each treatment were compared using L.S.D. test at 5%, according to Steel and Torrie (1980).

 

RESULTS

 

Regarding explant sources, the presented data in Tables (1 and 2) and Figure (2- A, B, C and D) indicate that there was no visible difference between shoot tips and leaf primordia derived from either aged tree of date palm or juvenile offshoot for their effect on survival percentage, while degree of browning was lower with M1 medium as compared to M2 medium after eight weeks of incubation. Although, explants of offshoot or aged tree significantly showed an increase in growth value when cultured on M1 medium compared to the same explants on M2 medium, but explants of offshoot observed higher growth value than explants of aged tree when both cultured on M1 medium. However, results in Tables (1and 2) confirmed the superiority of M1 medium which, decreased oxidative browning, prevented callus formation and stimulated growth of all tested explants. While M2 medium which contained 100 mg/L 2,4-D and 3 mg/L 2iP stimulated all explant tissues to form callus  with a significant lower growth value compared to M1medium.

          Response of aged tree explants showed some biological differences on M2 medium, as degree of browning, growth value and percentages of callus formation recorded 2.67, 85.3 and 22.22 % respectively for shoot tip and 1.56, 71.4 and 44.44 for leaf primordial. Shoot tip explants derived from juvenile offshoot on the same medium (M2) recorded 1.78, 30.1 and 33.33% for degree of browning, growth value and percentage of callus formation, respectively and 1.33, 59.9 and 66.67%, respectively for leaf primordial.

 

Table 1: Responses obtained from various explant sources of date palm c.v Samany cultured in  vitro on two types of media for 8 weeks, (survival percentage and degree of browning)

Explant sources	Explant types	Survival  %			Degree of browning
		Medium         (M1)*	Medium (M2)**	Mean (A)	Medium (M1)*	Medium (M2)**	Mean (A)
Aged trees	Shoot tip	100	100	100	1.0 (b)	2.67 (a)	1.84 (a)
	Leaf primordia	100	100	100	1.0 (b)	1.56 (ab)	1.28 (a)
Offshoots	Shoot tip	100	100	100	1.0 (b)	1.78 (ab)	1.39 (a)
	Leaf primordia	100	100	100	1.0  (b)	1.33 (b)	1.17 (a)
Mean     (B)		100	100		1.0 (b)	1.83 (a)

Means within each column followed by the same letters,were not significantly different at P= 0.05 according to the LSD test.

Rating Scale:  No browning (1),   Average browning (2),            High browning (3)

 (M1)*: MS + NOA (5 mg/L) + NAA (5 mg/L) + Kin (3 mg/L) + 2iP (3 mg/L)

 (M2)**: MS + 2,4- D (100 mg/L) + 2iP (3 mg/L)

Table 2: Responses obtained from various explant sources of date palm c.v Samany  cultured in vitro on two types of media for 8 weeks, (growth value and callus formation).

Explant sources	Explant types	Growth value			Callus formation  %
		Medium (M1)*	Medium (M2)**	Mean (A)	Medium (M1)*	Medium (M2)**	Mean (A)
Aged trees	Shoot tip	78.9 (d)	85.3 (c)	82.10 (a)	00.0 (e)	22.22 (d)	11.11 (d)
	Leaf primordia	91.0 (b)	71.4 (e)	81.20 (a)	00.0(e)	44.44 (b)	22.22 (b)
Offshoots	Shoot tip	85.3 (c)	30.1 (h)	57.70 (c)	00.0(e)	33.33 (c)	16.66 (c)
	Leaf primordia	95.5 (a)	59.9 (f)	77.70 (b)	00.0(e)	66.67 (a)	33.33 (a)
Mean    (B)		87.68( a)	61.68 (b)		00.0 (b)	41.66 (a)

Means within each column followed by the same letter are not significantly different at P= 0.05 according  to the LSD test.

Rating Scale:  No browning (1),   Average browning (2),        High browning (3)

 (M1)* : MS + NOA (5 mg/L) + NAA (5 mg/L) + Kin (3 mg/L) + 2iP (3 mg/L)

 (M2)**: MS + 2,4, D (100 mg/L) + 2iP (3 mg/L)

 

Results in Table (3) and Figure (2- F, G and H) show the effect of M1 and M2 media on inducing variable responses from shoot tip and leaf primordial explants of both aged tree and offshoot of date palm. M1 medium recorded beneficial effect in enhancement direct organogenesis and direct embryogenesis from leaf primordial explants 11.11 and 33.33 % of aged trees and 22.22 and44.44 % for juvenile offshoots respectively as compared to M2 medium which enhanced callus formation and subsequently indirect embryogenesis only in all tested explants. Interestingly, regardless type of medium or explant source, shoot tip explants did not show any direct organogenesis or embryogenesis within 4 months of culture incubation. Indirect somatic embryos regenerated from callus later on were subjected to rooting stage.

         In Table (4), data on the main effect of source and type of explants reveal that, leaf primordia of aged tree significantly observed the highest percentage of rooting (72.22%) followed by the treatment of leaf primordia of offshoot (68.52%). As for the main effect of incubation time, data show that highest root percentage was significantly observed at the third record (after 3 months) with charcoal.  Regardless charcoal, the gradual increase in incubation time up to three months resulted in a gradual increase in rooting percentage in the two types of explants (shoot tip and leaf primordium) of both sources (aged tree and offshoot). Concerning the interaction, all explants significantly showed

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

higher response after 3 months, except the treatment contained shoot tip of aged tree in the medium of charcoal.

           Data presented in Table (5), show the effect of soil type on the survival of plantlets produced from shoot tip and leaf primordial of both aged tree and juvenile offshoot. Data on the main effect of soil type clear that the highest percentage of survival (64.85%) was obtained with the mixture of peatmoss + vermiculite {1:1, v/v, (Figure, 2 J)} followed by the mixture of peatmoss + perlite (1: 1, v/v) that observed 61.15% survived plantlets. As for the main effect of source and type of explants, data show that with both aged tree and offshoot, leaf primordia were significantly effective in increasing the percentage of survival compared to shoot tips. Concerning the interaction, peatmoss + vermiculite recorded the highest percentage of survival with plantlets derived from all tested explnts.

               

 Table 5: Effect of plantlet sources and soil types on survival percentage date palm c.v Samany after 3 months ex vitro in acclimatization stage.

Means within each column followed by the same letter are not significantly (N.S) different at P= 0.05 according   to the LSD test.

        

In Table (6), data on the main effect of soil type on growth vigor reveal that, the soil mixture contained peatmoss + vermiculite (1:1, v/v) also significantly enhanced the growth vigor (5.0) followed by the record 4.0 of the  mixture contained peatmoss + perlite (1 : 1, v/v). However, data on the main effect of both sources and types of explants show no significant difference between them. Interestingly, results of the interaction reveal that, peatmoss + vermiculite recorded the highest growth vigor of plantlets derived from all tested explnts.

 

Table 6:  Effect of plantlet sources and soil types on growth vigor of date palm c.v Samany in acclimatization stage.

Means within each column followed by the same letter are not significantly (N.S) different at P= 0.05 according   to the LSD test.

 

DISCUSSION

 

Cultured explants on M1 medium observed good survival percentage with no contamination or callus formation as well as low oxidative browning. After two to three subcultures of shoot tips and leaf primordia  at the same medium (M1), shoot tips enlarged, while leaf primordial observed bud growth and varying response to the production of direct somatic embryos at the base of the leaf primordial. Our results are in agreement with those obtained by Beauchesne et al., (1986) and Sudhersan et al., (1993) they found that, at the bottom of the young leaves, some very little axillary buds are often visible, normally after four to six months, the bottom of young leaves in cultures, gave some signs of budding which was indicator for giving true- to-type plantlets. Plantlets produced by the organogenesis process should be clonal and produced with less risk of genetic aberrance than callus-derived plantlets (Tisserat, 1981). Swelled shoot tips which were recultured on nutrient media contained low level of auxin 2-5 mg/L NAA, 3.0 mg/L kin and 3.0 mg/L 2ip observed shoot development and varying responses to multiplication. These results are in agreement with Tisserat (1979) who found that buds and tips cultured on medium contained low auxin concentrations initiated leaves and in some cases roots while, media contained high auxin concentrations resulted in the formation of callus from buds and tips. In addition to, Mater (1986) also observed that high auxin levels in the medium stimulated callus growth and low auxin levels favored normal vegetative growth of the shoot tip explants. Also, Thorpe (1980) suggested that organogenesis is a very complex physiological process to be regulated by hormonal balance alone. Moreover, Beauchesne et al. (1986) reported that beauchesne's medium supplemented by various auxins at low level concentration, enhanced bud growth in vitro. On the other hand, Zaid and Tisserat (1983) reported that, addition of growth regulators to nutrient medium was not necessary to stimulate shoot proliferation, better shoot tip development occurred on nutrient media that contained 10 and 100 mg/L NAA. In addition to, Tisserat (1984) who reported that the addition of cytokinin at any level to date palm tissue culture media did not enhance shoot differentiation. EI-Henawey et al. (1982) reported that root and shoot differentiation occurred on MS media supplemented with both NAA and kinetin. The present results indicated that leaf primordia showed varying responses to give buds and embryos, after sufficient growth of the leaf primordia and their buds, they were separated carefully and recultured. Somatic embryos as well as adventitious buds were transferred to hormone and activated charcoal-free medium for germination. Results are in agreement with those obtained by Tisserat (1979), Beauchesne et al., (1986) and Sudhersan et al. (1993) who found that hormone-free medium enhanced embryos production and development. Individual shoots showed good growth and elongation when subjected to hormone-free medium. About 15 cm long shootlets were rooted well when transferred to the MS basal medium supplemented with 0.2 mg/L NAA (Figure 2-I). Generally shootlets composed of two or more leaves with roots of 4.0 cm or longer length were better than smaller shootlets in acclimatization. We found that plastic pots which contained soil culture consists of peat moss and vermiculite in equal proportions (Figure 2-J) enhanced the survival of plantlets in the greenhouse within three months of acclimatization. In this regard, Ziv (1986) reported that about 50 to 90 % of in vitro propagated plantlets of many species have been lost at the time of transfer to soil. Our results are in agreement with Al-Jibouri et al. (1988) who found that, survival of date palm plantlets reached about 29, 76 and 88 % by using either of vermiculite or sphagnum peat or (1:1) mixture of both, respectively.

This study indicated that mass production of true- to - type plants from adult date palm is possible in vitro (Figure 1). This is could be the first report on direct embryogenesis of adult date palm according to the available literature.

Date palm explants

    Leaf                                                                                                                           Shoot

   primordia                                                                                                                   tip

    

                                                                                                                                     

                                                                                                

   

 

     Adventitious         Direct                                   Callus                                      Axillary   

          buds                embryos                                                                                           buds

 

 

                                                                             Indirect

                                                                               embryos

                                                                                                 

                                                                                                           

 

                                                                             Repetitive embryos

 

 

                                                                                                 

                                                                                                

 

 

 

 

 

                                                                Germination

                                                                                              

                                                                   Shootlets

                                                                                                

 

                                                                                          Rooting

                                                                                               

                                                                                           Plantlets

                                                                                          

                                                                              Greenhouse acclimatization

                                                                                           

                                                                                   Open filed transfer 

 

              Figure 1: Date palm propagation through tissue culture techniques.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure (2): Date palm c.v Samany in micropropagation stages:-

     A –Aged tree after isolation             F- Emvrtogenic callus regenerated indirect embryo

     B –Offshoots ready for separation     G- Leaf primordia regenerated direct embryos

     C –Mother tissue in sterilization process.  H- Leaf primordia gave adventitious buds

     D –Shoot tip explants after excision           I- Shootlets on rooting medium + charcoal

     E – Leaf primordial explant after separation. J- Plantlets in peatmoss + vermiculite

Conclusively, using explants from aged trees or juvenile offshoots forin vitro production of true to type date palm plantlets was carried out in this study through direct organogenesis or embryogenesis (avoiding callus formation). That was achieved when leaf primordia of both sources were cultured on medium of Hegazy (2003) for 4 months. The obtained embryos or buds were grown and developed on MS hormone-free medium, and then, they transferred into MS basal medium contained NAA (0.2 mg/L) asrooting medium for 3 months. Plantlets were acclimatized in soil mixture contained peatmoss and vermiculite (1: 1, v/v).  

 

 

REFERENCES

 

Al-Jibouri, A. J. M.; Salman, R. M.  and Omar, M. S. (1988). Transfer of in vitro regenerated date palms to the soil. Date Palm J., 6 (2):390-400.

Beauchesne, G.; Zaid, A. and Rhiss, A. (1986). Meristematic   potentials of bottom of young leaves to rapidly propagate date palm. Proceeding of the Second  Symposium on Date Palm, King Faisal University , Al-Hassa , Saudia Arabia , pp. 87- 93.

Drira, N. (1983). Vegetative propagation of date palm (Phoenix dactylifera L.) by in vitro culture of axillary buds and of leaves. Compters Rendus des seances de L  Academie des sciences, III sciences de La vie. 296 (22): 1077-1082.

EI-Henawey, H. M., Bondok, A. E., Habib, S. A. and Sabour, A. M.  (1982): Trails on vegetative propagation of date palm by means of tissue culture. Proceedings of the first symposium on date palm. King Faisal University, Al- Hassa, Saudi Arabia, pp. 702-704.

Hegazy, A. E. (2003): Some physiological studies on date palm micropropagation through direct somatic embryogenesis. Ph. D. Thesis. Plant Physiol. Dep. Fac. of Agri. Cairo Univ. Egypt. 

Mater, A. (1986). In vitro regeneration of ( Phoenix dactylifera L.). Date Palm J., 4 (2): 137-152.

Murashige, T. and Skoog, F. A. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol., 15 : 433-479.

Phillips, G. C. and Hubstenberger, J. F. (1995). Micropropagation by proliferation of axillary buds. In: Plant Cell, Tissue and Organ Culture, Fundamental Methods (Gamborg, O. L. and Phillips, G. C., eds). Springer-Verlag Berlin Heidelberg, pp. 45-54.

Pottino, B. G. (1981). Methods in Plant Tissue Culture. Dept. of Hort., Agric., Maryland Univ., College Park, Maryland , USA, pp. 8-29.

SAS (1988): Statistical analysis system SAS User’s Guide: Statistics SAS        Institute Inc., Cary, N. S., ed.

Steel, R. G. and Torrie,  J. H. (1980).  Principles and Procedures of Statistics, a Biomerical Approach. Mc Grow- Hill Book Company, New York, 469-517.

Sudhersan, C., Abo El- Nil, M. M. and Al- Baiz, A. (1993): Occurrence of direct somatic embryogenesis on the sword leaf of in vitro plantlets of (Phoenix dactylifera L.) cultivar Barhee. Current scince 65: 887- 888.

Thorpe, T. A. (1980): Organogenesis in vitro: structural, physiological and biochemical aspects. Int. Rev. Cytol. Suppl. 11A:71-111.

Tisserat, B. (1979). Propagation of date palm (Phoenix dactylifera L.) in vitro. J. Exp. Bot., 30 (119): 1275-1283.

Tisserat, B. (1981). Production of free- living date palm through tissue culture. Date   palm Journal 1: 43 – 54.

Tisserat, B. (1984). Tissue culture of date palm. Hand Book of Plant Cell Culture, 2: 505-545.

Veramendi, J. and Navarro, L. (1996). Influence of physical conditions of nutrient medium and sucrose on somatic embryogenesis of date palm. Plant Cell Tiss. Org. Cult., 45: 159-164.

Zaid, A. & De Wet, P.(2005): Date Palm Cultivation. FAO publication, within the framework of the date production Support program, Vol.           156.

Zaid, A. and Tisserat, B. (1983). In vitro shoot tip differentiation in (Pheonix dactylifera L.). Date Palm J., 2 (2): 163-182.

Ziv,  M. (1986):  In vitro hardening and acclimatization of tissue culture plants. In : lant Tissue Culture and its Agriculture Application, (Withers L.A., Alderson,  P.G., eds.) Butter Worths, London . pp. 187-196.

Ziv, M. (1992): The use of growth retardants for the regulation and acclimatization of in vitro plants. In: Karssen, C.M.; Van loon, L.C. and Vreugdenhil, D. (eds). Progress in Plant Growth Regulation, pp. 809-817

 

 

 

 

 

استجابة المنفصلات النباتیة لنخیل البلح المسن والخلفات خلال الإکثار الدقیق

 

 

حمدی احمد عماره

معهد الهندسة الوراثیة والتکنولوجیا الحیویة – جامعه المنوفیة- ج.م.ع

 

أجریت هذه الدراسة فی معمل زراعه الأنسجة النباتیة بمعهد الهندسة الو راثیة خلال الفترة من 2004-2006 .تهدف هذه الدراسة إلی معرفه إمکانیة إکثار نخیل البلح المسن صنف سمانی معملیا ومقارنته بإکثار الخلفات تحت نفس الظروف. وقد استخدم کل من البادئات الورقیه والقمم النامیة المفصولة من نخیل مسن وخلفات صغیره للأکثار معملیا وذلک بزراعتها علی نوعین من البیئة المغذیة{ حجازی ((M1 وتیسیرات ((M2 } وقد أظهرت النتائج انه لا یوجد أی فرق معنوی بین تأثیر نوعی البیئة تحت الدراسة علی حیویة کل المنفصلات المستخدمة, إلا انه وجد أن نسبه التلون باللون البنی  سجلت أقل معدلتها عند زراعه کل المنفصلات النباتیة علی بیئة حجازی وذلک مقارنة ببیئة تیسیرات , وقد سجلت النتائج ان جمیع المنفصلات النباتیة أدت إلی تکوین کالس عند زراعتها علی بیئة تیسیرات وذلک بعد 8 أسابیع وکذلک بعد 4 شهور من الزراعة وذلک مقارنة ببیئة حجازی التی لم یظهر عند استخدامها أی ظهور للکالس . وجدیر بالملاحظة أن البادئات الورقیه فقط لکل من الأشجار المسنه والخلفات قد شجعت علی تکوین أعضاء خضریه واجنه جسدیه مباشرة بدون تکوین کالس عند زراعتها علی بیئة حجازی لمده  4 شهور . کما وجد أن القمم النامیة للأشجار المسنه والخلفات لم تظهر أی إستجابه مباشره لتکوین الأعضاء والأجنة .وقد استکملت البراعم والأجنة نموها وتطورها علی بیئة حجازی بدون منظمات نمو ثم نقلت علی بیئة تجذیر عبارة عن موراشیج وسکوج المحتویة علی 0.2 ملجم \ لتر نفثالین حامض الخلیک  وقد اجتازت النباتات الناتجة مرحله الأقلمة عند زراعتها فی خلیط من بیئة الزراعة المحتوی علی بیتموس وفرموکلیت بنسبه 1:1 (بالحجم).

 

 

 

 

 

 

 

 

 

 

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