Science International  Volume 6 Issue 1, 2018

Review Article

Factor Controlling Micropropagation of Fruit Trees: A Review
S.A.M. Hassan
Biotechnology Lab., Department of Pomology, National Research Centre, Postal Code 12622, Dokki, Giza, Egypt

Nagwa S. Zayed
Biotechnology Lab., Department of Pomology, National Research Centre, Postal Code 12622, Dokki, Giza, Egypt

ABSTRACT:
There were several factors controlling micropropagation of fruit trees such as explant type and size, surface sterilization , phenol exudation and its control, different culture medium, different media (strength, type and state), carbon source and additives, light and temperature, plant growth regulators, pH and agriculture media. The data indicated that smaller explants 0.5 mm such as shoot tip culture lead to establishment stage of in vitro culture which will be free from viruses free of the most plant species. The best surface sterilization was obtained when the explants were sterilized by using (10-30%) clorox solution with two drops of tween 20 for 15-20 min. According to explant type (shoot tip, axillary bud, one node cutting, leaf disc, flower buds and seeds). The incorporations of antioxidant agent (150 mg L–1 citric acid+100 mg L–1 ascorbic acid) for 2-24 h prior culturing in the medium is the most treatments for controlling phenol exudation. The pH of the media was adjusted to 5.7 gave better response in growth and proliferation of explant and induction of roots in many explant species. The cultured explants were incubated under 16 h of artificial light and 8 h of darkness at average temperature of 25-28°C in several explant species. Sucrose is the most common carbon source and energy because sugar is reported as a source of energy and carbon in inducing growth and development during all different stages of micropropagation. Culture at full medium strength supported better growth and proliferation in most explant species. Also, liquid medium is the best medium state during in vitro establishment stage when the phenolic products released from culture explants such as (Date palm, Mango, apple and some explant types) and rooting stage. The plant growth regulators such as (auxins, cytokinins and gibberellins) are required in very minute quantities. Both BAP and 2ip have been preferred for used in vitro proliferation media for most fruit micropropagation. In addition, NAA and IAB are the least stable in vitro rooting media. Also, gibberellins are used less frequently compared to auxin and cytokinins. Using combination of agriculture media consists of vermiculite: sand: peatmoss (1:1:1) is the best suitable media during in vivo acclimatization stage in the most plantlets.
 
    How to Cite:
S.A.M. Hassan and Nagwa S. Zayed , 2018. Factor Controlling Micropropagation of Fruit Trees: A Review. Science International, 6: 1-10
DOI: 10.17311/sciintl.2018.1.10
 


INTRODUCTION

Micropropagation is one from tissue culture which allows the production of large number of plants from small pieces of the mother plant in relatively short period of time and limited space. Micropropagation as recent technique is mainly used for bulk rapid propagation of several commercial plant species included date palm1. Micropropagation technology offers mass propagation and clean planting material. Micropropagation has the primary advantage of producing a disease which can be solved by propagation of banana2. Tissue culture micropropagation has been employed to aid in the clonal propagation of numerous plant species. Tissue culture technique may offer a possible method to produce a large number of genetically uniform palms identical to other plants and normal fruit after 4 years from planting and production of date palm plants free from diseased, almost 100% survival rate compared with vegetative of shoots due to the presence of a strong root system on them1. Surface sterilization is the most important step in preparation of explants for in vitro because controlling bacteria and fungi contamination of most fruit plants from field sources is very different3. The successful applications on micropropagation of date palm during in vitro acclimatized depending on the appearance of number of leaves before transplanting in the greenhouse4. Thus, the ultimate goal of this review was to find out the factors controlling micropropagation of fruit trees.

Mother plant (explants type, genotype and explants age): The highest shoot number and optimal shoot regeneration were obtained when cultured on MS medium supplemented with 6.0 mg L–1 BA and 0.1 mg L–1 NAA on leaf sections of pyrus communism5. A protocol for in micro propagation of mature plants of citrus limon using nodal shoot segments6. The problem of phenolic exudation and contamination of guava c.v. "Banaras" was not observed when somatic embryo derived young and aseptic plantlets were used as explant source7.

Using shoot tips of three fig cultivars (Aboudi, Gizy and Sultany) plantlets from the establishment stage were cultured individually on Murashige and Skoog (MS) medium supplemented with 0.5 mg L–1 6-Benzylaminopurine were increasing explants development and reducing necrosis and browning8. In the same time, Emam9 found that shoot tips of pear rootstock was successfully developed when cultured on MS medium as compared with one-nodal cutting. Strosse et al.10 concluded that shoot tips from young suckers in banana was successfully developed when cultured on MS medium during the establishment stage. Also, Sutherland et al.11 found that shoot tips explants in banana were superior increasing proliferation of homogenous and diseases free plantlets. Whereas, Baiea12 found that leaf discs explants of two peach rootstocks were successfully developed when cultured on MS medium (Fig. 1).

The best number of shoots (5.35 shoots/responding explant) of Jatropha curcas explants occurred from axillary buds13. Similarity shoot tip, stem and axillary bud explants were successfully used for micropropagation of Jatropha Curcas14. In the same, Sumalatha15 Found that shoot tip as the explant was successfully used for micropropagation of banana plants. The best shoot proliferation of Jojoba distinguished clone from shoot tip and nodal segments16.

The nodal segments as the explants were successfully used for callus induction as compared to leaf and root segments of citrus plants17. In the same time, somatic embryo derived young and aseptic plantlets were used as explants source were successfully used for micropropagation of guava c.v."Banarasi"7.

The same time, the longest roots were obtained when the c.v. Golden delicious control and by genotype 4566 were cultured on WPM medium as compared with the genotypes 4608, Gi47 7/4 and C/90718.

Surface sterilization of explants: Banana plants were surface disinfected by 0.1% HgCl2 solution containing tween 20 for 5 min and washed thoroughly with sterile, deionized water15.

Meanwhile, sterilize shoot tip and nodal bud using sodium hypchlorite Clorox (NaOCl) for 20 min which gave good explants survival in pomegranate19. Using a combination of NaOCl and Na methiolate for 20 min are the two most successfully sterilize axially bud and segments in pomegranate which gave good explant survival (65%)20. In the same time that using NaOCl (0.75%) gave the maximum survival percentage (46.66%) followed by NaOCl (1.0%) with 26.66% survival percentage as compared with NaOCl (1.25%) with the minimum survival (6.6%) of Jack fruit21 .

In another study during trials on in vitro propagation of mangoes, using combination of 10% sodium hypochlorite "Clorox" (sodium hypochlorite 5.25%) NaOCl and 0.05% mercuric chloride (HgCl2) for dipping durations 7 and 10 min give the highest survival percentages and lower contamination percentage shown in Fig. 2. Also, using 10% sodium hypochlorite was successful in surface sterilization and gave good explant in very low visual contamination of rootstocks Mariana (Prunus mariana)22.

In addition, surface sterilization gave good explanation for grape tissue (Vitis vinifera) successfully by using combination of 10% sodium hypochlorite and surfactant drops for 10 min23. Three doses, 0.05, 0.1 and 0.2% of aqueous sodium hypochlorite or mercuric chloride solution separately for 10 min were used as surface sterilize.

Figure 1: Different types of explant

Figure 2: Different surface sterilization of Jojoba

It was found that using 0.1-0.2% mercuric chloride increased aseptic culture establishment but inhibited by break due to toxicity to explant of Bambusa tulda24. In the same time dipping explants in 15% of sodium hypochlorite for 15 min was effective in sterilizing grape root stocks25.

Phenol exudation and its control: It was mentioned that in Fig. 3a, b, using activated charcoal in the cultural medium induced initiation proliferation of woody plant but the negative effect of activated charcoal by adsorbing growth regulators and lowering pH of the medium26. In the same time dark treatment for few days of initiated shoots reduces the release of phenolic compounds from the shoots of apple root stocks27.

Figure 3(a-b): Effect of anti oxidant on phenol oxidation of date palm, (a) Control and (b) Antioxidant solution 150 mg L–1 Citric + 100 g L–1 ascorbic

Similarly attempted in vitro culture of pomegranate c.v. Mridula, nodal segments with sterile wax decreased the phenol exudation and lead to good explant percentage of cultural establishment28. Using ascorbic acid was the most successfully method to reduce oxidation during the establishment stage of apple18.

Different methods such as keeping explants in dark after culture, adding antioxidants such as citric acid (100 mg L–1) and ascorbic acid (150 mg L–1) for 30 min to the medium as well as adding activated charcoal (3 g L–1) to the medium. These treatments reduce phenolic compound production and browning of olive explants29.

Medium (type, strength and state): It was reported that full strength of Murashige and Skoog medium is more suitable for establishment of coffee plantlets30. Number of leaves and shoot length of fig plants was induced on double strength Murashige and Skoog medium showing in Fig. 4(a-d). Meanwhile, the highest value of shoot number was obtained on half MS strength medium31. Also, Mukherjee et al.32 mentioned that shoot proliferation from nodal explants of grape root stock was induced on half strength Murashige and Skoog medium.

Figure 4: Effect of medium strength of sultany fig cultivar, (a) Full strength, (b) Double strength, (C) One-half and (d) One-quarter

Figure 5: Effect of liquid media on apple rootstock

On the highest proliferation rates and shoot growth of apple as shown in Fig. 5 were also obtained with MS medium replacing Fe-EDTA by Fe-EDDHA as iron source. In the same case, woody plant medium (WPM) is the most important to avoid contamination by fungi and bacteria as well as phenolic oxidation during the establishment stage18.

Similarly the highest shoot proliferation on Citrus Limon was obtained with MS medium with lower concentrations of ammonium and potassium nitrates6.

On the other hand, used 3/4 medium strength was the most suitable medium for development of Jojoba clones16. acclimatization of plantlets derived from tissue culture confirmed the efficiency and importance of this method. The subculture of rooted shoots into MS salts solution and increasing light intensity enhanced the plantlet photosynthesis and then changing it from heterotrophic to autotrophic status of date palm33.

The highest survival percentage (70-86%) of date palm was obtained on half medium strength after 1 month pre acclimatization stage as compared with the direct transfer to the greenhouse ranging from 12-28%, Mazri34. Also, one-half strength MS medium was the most suitable medium for the maximum number of roots and root length of almond plantlets, c.v. Nonpareil35.

The highest rooting with higher number of roots (10.33 roots/shoot) per micro shoot found on half strength MS basal medium in pomegranate19. was obtained36 found that the best significant improvement of greening and explants development per micro flower bud on full and one-half medium strength compared with one-quarter medium strength of pear "Le Conte" flower bud.

In respect that several factors affecting on in vitro root formation of date palm c.v. Boufeggous plantlets such as with solid or liquid medium just before acclimatization37. While, the used of liquid medium is more suitable for medium state (solid or semisolid media) before the transfer of plantlets to the greenhouse of Barhee date palm38. Also, use of a liquid medium before the transfer of plantlets to the greenhouse of date palm c.v. Najda was very effective in improve shoot quality34. The highest survival percentage of in vitro acclimatized date palm c.v. Barhee was recorded with plantlets growing on liquid Ms medium compared with the other medium status39.

The highest rooting percentages on apple was observed when the plantlets were cultured in liquid organized medium supplemented with IBA at 25 μM on all genotypes including M. domestic c.v Golden delicious18.

Supporting media: The best number of shoots and shoot length in pineapple plantlets were induced to form shoots on MS medium containing 2 g L–1 gelrite. Meanwhile, an agar at the rate of 7 g L–1 was better than gelrite during multiplication stage40. Similar , increased response with the decrease in agar from 0.8-0.4% in the medium improve the in vitro root and shoot parameters as compared with the other supporting structures banana (musa spp) c.v. Grand naine plantlets41. Also, added to the media with gelling specialist agar at 5-8 g L–1 in the medium were successfully used for microprogation of banana plants15.

Carbon source and additive: It was revealed that medium supplementation with 30 g L–1 sucrose gave the best result of shoot tip explants of papaya42. Similarly, sucrose at the rate of 30 g L–1 concentration as carbon source was proved to be best resolute on in vitro rooting of banana (Musa spp) c.v Grand naine plantlets41.

In the same time using of MS medium supplemented with 20 g L–1 fructose induced shoot proliferation of the two fig cultivars (Sultany and Aboudi) as compared with the explants an media without sucrose shown in Fig. 6(a-d)31.

In the respect, addition of both fructose and glucose to Murashige and Skoog medium resulted in improving shoot length and leaf number of jojoba plants. While, sucrose proved to be the best carbon source for shoot number16. On the same time, supplemented the culture medium with 30-60 g L–1 sucrose encouraged maximum explant development and shoot growth of date palm while fructose produced the highest values of dry weights as compared with other carbon sources treatments such as glucose, sucrose and maltose43.

The same addition also, one-half medium strength treatment enhanced the proliferation percentage and shoot number compared with the other treatments36.

Whereas, addition of either adenine sulphate (80 mg L–1) or yeast extract (300 mg L–1) was effective in highest callus production and reduced necrosis and browning of both Nemagaurd and Okinawa root stock12. The same, olive medium (OM) supplementation with 50 ml L–1 coconut water gave the best result of the highest proliferation rates with an average of 3.4 new explants on each 30 days in olive44. While used sucrose concentrations at the rates of (30-40 g L–1) as a carbon sources gave the best results for microprogagation of banana pants15.

Light, temperature conditions and pH requirements: In vitro plantlets of date palm c.v. Barhi are generally grown under 2000 lux, through using different numbers of white cool fluorescent lamps for 16 h of light and 8 h of dark, improved most parameters under study such as root length, root number, shoot length and greening as compared with the other light intensities. Meanwhile, increasing shoot thickness was recorded when the cultures were subjected to light intensity of 3000 lux4. In vitro plantlets of date palm are generally grown under high light intensity enhanced the plantlet photosynthesis33.

Similarly, tasted in vitro plantlets under high light intensity (4000-12000 lux) and temperature (26-36°C) might cause charring of leaves and wilting of plantlets45. Also, in vitro root formation of date palm c.v. Barhi, the best results for shoot thickness was recorded when the cultures were subjected to a light intensity of 3000 lux . However, number of root, root length, number of leaves and greening were recorded when the cultures were subjected to light intensity of 2000 lux39.

Figure 6: Effect of carbon source of Aboudy Fig. cultivar, (a) Glucose, (b) Fructose, (c) Sorbitol and (d) Sucrose

The same result found that several factors affecting the in vitro root formation of most fruit trees, the best results for both root number and root length were recorded when the culture were subjected to light intensity of (4.000-12000 lux) and temperature (26-36°C) might cause charring of leaves and increasing of plantlets45. While, during acclimatization plantlets of date palm, increasing light intensity increased the plantlets photosynthesis33.

On the other hand in vitro plantlets of banana were generally grown under temperature 28°C and presented of light to 12-16 h15. Also, cold pretreatment of explant for 3 days at 5°C encouraged the best responses of peach root stocks12.

The best root and minimum time for root initiation with longest length at PH 5.5 during the preparation of the medium on in vitro rooting of Banana plantlets c.v. Grand naine were obtained by Ahmed et al.41.

Plant growth regulator: The supplementation of the culture medium with BA (0.5 mg L–1) and IBA (0.25 mg L–1) induced the highest number of shoots after 30 days of culture on Jatropha Curcas14. The supplementation of the subculture medium with 0.5 mg L–1 BA and 0.5 mg L–1 IBA were necessary for differentiation of the shoot bud as shown in Fig. 7a-c. While only 1.0 mg L–1 IBA induced the shoot elongation of Jatropha Curcas46. similarly, the supplementation of the culture medium with 6.00 mg L–1 IBA induced the best multiplication of coffee plantlets, in the some cases, number of roots and root length/plantlets were greatest with 3.0 g L–1 IBA30.

In another study the supplementation of the culture medium with 3 mg L–1 BA and 0.5 mg L–1 NAA induced the regeneration response (71.89%) from model segment in citrus plants. In some cases, maximum rooting percentage (71%) was obtained with modal segment derived callus cultured on Ms medium supplemented with 0.5 mg L–1 NAA and 3 mg L–1 BA17. While the supplementation of the culture medium with 2.0 mg L–1 BA and 0.0, 0.5 or 1.0 IBA mg L–1 induced the best number of regenerated plantlets of either Nemagaurd or Okinawa rootstock12.

The same supplementation of the culture Ms medium with 1.0 mg L–1 BA induced the best multiplication of almond plantlets c.v. Nonpareil. In the same cases, number of roots and root length/plantlets was greatest with 8.0 mg L–1 indole acetic acid35.

Similarly, the supplementation of the culture medium with 1.0 mg L–1 BAP combined with 1.0 mg IBA was the best for in vitro culture initiation of apple plantlets27. Also, the supplementation of the culture medium with 1.0 and 1.5 mg L–1 BAP combined with 1.0 mg L–1 IBA induced the survival percentage (96.7%) of MM 106 and (93.3%) Anna apple plantlets47.

In addition, supplementation of Ms medium with 0.5 mg L–1 BAP induced the shoot proliferation (5.37 shoots/explant) of a dwarfing cherry root stock48. Similar results recorded that the supplementation of the Olive medium with 2.22 mg L–1 BAP induced the best proliferation rates with average of 3.4 new explants after 30 days. In the same cases, rooting percentage 85% was obtained by OM with 3 g L–1 IBA,44. In the same time, the highly cytokinin induced the best shoot multiplication of jojoba.

Figure 7: Effect of growth regulators, (a) BAP, (b) IBA and (c) Agriculture media

Figure 8: Effect of agricultural media Pomegranate

Also, callus induction was greatest with Auxins alone or combined with cytokinin16.

Cytokinin 6 benzyl aminopyrine (BAP) has been found essential for obtaining good shoot proliferation in pomegranate. In the same cases, Murashige and Skoog (MS) medium containing 2.0 mg L–1 IBA has been found essential for obtaining good rooting during in vitro rooting19. Highest frequency of shoot proliferation was obtained in culture medium Quorin and lepoivre medium supplemented with BA at the rate of 0.4 mg L–1 and IBA 0.05 mg L–1 20.

Also, the highest rooting percentages (70.37) with dual auxin i.e. IBA and NAA supplemented to the rooting medium28 found that the highest rooting percentages (93.33%) and longer roots (3.29 cm) were obtained in culture medium added 0.5 mg L–1 IBA. In the same cases, high frequency shoot elongation was also obtained with MS medium supplemented with higher concentration of cherry laurel prunus laurocerasus L49.

Moreover, high frequency shoot proliferation was obtained on the basal medium supplemented with lower concentrations of BAP of sweet cherry cultivar "Lapins". On the other hand, good shoot elongation was also obtained with a higher concentration of BAP50. Increased shoot proliferation of olive was also obtained with increasing BAP concentration up to 2.1 mg L–1 in the media. The effect of high concentrations of BAP may be prevented by supplemented GA3 to the culture media during proliferation stage29. The highest shoots and number of shoots (4.66) per proliferated explant were obtained on MS culture medium supplemented with BAP at the rate of 1.5 mg L–1) of Jack fruit21.

Agricultural media: Data in Fig. 8, the highest survival percentages of plantlets, plant height (cm), plant thickness (cm) and number of leaves/plant occurred when agricultural media during acclimatization consists of a mixture of sand and peat moss (1: 1) was used of coffee plantlets30. Also, date palm c.v. Barhee plantlets were successfully acclimatized when transferred to pots containing a mixture of (25% vermiculite+50% and + 25% peat moss)39. While, rooted shoots (plantlets) were transplanted in small pots containing a mixture of peat moss and perlite (2:1) and placed in plastic tunnels or in a greenhouse. The survival percentage was 85% after 3 months when the plants were transferred to bigger pots of Maktom c.v. Date palm33.

In addition, the highest survival percentage (95%) of olive plantlets were obtained during acclimatized when transferred to pots containing a mixture of vermiculite: Perlite 3: 1 (V/V) substrate44. In the same cases, the highest survival percentage (85%) after 3 months of date palm c.v. Maktom plantlets during acclimatization stage when transplanted in a small pots containing a mixture of peat moss and perlite (2: 1) and placed in plastic tunnels in a greenhouse33.


CONCLUSION

This study indicated that smaller explants 0.5 mm such as shoot tip culture will be free from viruses free of the most plant species. The incorporations of antioxidant agent prior culturing in the medium is the most treatments for controlling phenol exudation. The pH of the media was adjusted to 5.7 gave better response in growth and proliferation of explant and induction of roots in many explant species. Sugar is reported as a source of energy and carbon in inducing growth and development during of micropropagation. Culture at full medium strength supported better growth and proliferation in most explant species. The plant growth regulators such as (auxins, cytokinins and gibberellins) are required in very minute quantities.


SIGNIFICANCE STATEMENTS

This study discovers the factor that controls the micropropagation that can be beneficial for in vitro propagation of fruit tree. This study will help the researcher to uncover the critical areas of success of culturing explant in vitro culture and low cost process of propagation that many researchers were not able to explore. Thus a new theory on how to overcome many factors that are effective on explant success may be arrived at.


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