ENHANCED PRODUCTION OF ALOE MANNAN AND OTHER CONSTITUENTS USING PLANT BIOTECHNOLOGY
Thesis submitted for The Fulfillment of the Degree of DOCTOR OF PHILOSOPHY by ZARREEN BADAR
DEPARTMENT OF BIOTECHNOLOGY UNIVERSITY OF KARACHI KARACHI-75270, PAKISTAN 2014
DECLARATION
I hereby declare that this thesis entitled, “ENHANCED PRODUCTION OF ALOE
MANNAN AND OTHER CONSTITUENTS USING PLANT BIOTECHNOLOGY” submitted to the University of Karachi has not been submitted elsewhere for the fulfillment of Ph.D. or any other dissertation.
ZARREEN BADAR
Department of Biotechnology,
University of Karachi
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CERTIFICATION
I have gone through the thesis entitled, “ENHANCED PRODUCTION OF ALOE
MANNAN AND OTHER CONSTITUENTS USING PLANT BIOTECHNOLOGY” submitted by Mrs. Zarreen Badar for the award of Ph.D. degree and certify that to the best of my knowledge it contains no plagiarized material.
Seal & Signature of Supervisor: ______
Name: ______
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DEDICATION
This dissertation is dedicated to my entire Family for always giving me faith, hope and confidence. Their unconditional love, sincere motivation, immense support and spiritual prayers have made this dream become reality.
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ABSTRACT (English)
Plants have been an exemplary source of medicine since ancient times. Today a wide array of active principles derived from plants represent numerous chemical drugs.
About 40% of compounds used in pharmaceutical industry are directly or indirectly derived from plants because the chemical synthesis of such compounds is either not possible and/or economically not viable. Aloe vera (Aloe barbadensis) is a tropical and subtropical plant with immense therapeutic potential. Aloe fresh gel, juice or formulated products have been used for medicinal and cosmetic purposes since centuries. Acetylated mannan also known as Aloe mannan is a storage polysaccharide in the pulp, the clear inner portion of leaf. The isolation of this unique bioactive compound “Aloe mannan” has drawn prime attention of many research workers. Chemical synthesis of Aloe mannan and other Aloe vera based beneficial products of pharmaceutical and medicinal value is difficult and expensive. Plant tissue culture is a promising technology, especially for the multiplication and production of novel and improved plants species and for an increased biosynthesis of products of industrial and medicinal value from vegetative resource.
MIcropropagation Studies: In the present study an elite Aloe barbadensis variety, having high acemannan levels was selected and the potential of indirect regeneration following an initial callus growth was explored. Maximum calli were induced under the dark on MS (Murashige and Skoog) medium supplemented with 2-8 mg/L of NAA (α- naphthaleneacetic acid), as compared to light treatment. Shoot regeneration was achieved at the highest frequency by sub-culturing of the calli on MS medium, containing 0.1 mg/L of IBA (Indole-3-butyric acid) and 1.0 mg/L of BAP (6-Benzyl
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amino purine), in light conditions. The plants rooted well on the same regeneration medium and acclimatized successfully under green house conditions. In other series of experiments, callus cultures were subjected to different stress conditions to investigate the potential of in vitro genetic variation, leading to the enhanced production of the immunomodulatory polysaccharide acemannan in plant calli and in vitro regenerated plants from callus tissues. Aloe vera calli were cultured in vitro with four different carbon sources (sucrose, mannose, glucose and galactose) and five concentrations (2, 3, 4, 5 and 6%) and kept in dark conditions for 1 month. Then the cultures were analyzed for acemannan content, which was found highest in the medium containing 3% mannose sugar. Thus according to the results achieved, it was evident that production of the bioactive polysaccharide acemannan was enhanced in the in vitro regenerated Aloe vera plants as compared to conventionally propagated plants in field. FT-IR, ¹H-NMR and HPLC techniques were employed for qualitative and quantitative analysis of this medicinal compound.
Acemannan Enhancement: The development of a callus route was availed to investigate the in vitro plants for enhanced production of the bioactive polysaccharide acemannan. The production media and light conditions played a vital role to increase the acemannan levels in stress conditions. The investigation revealed quite interesting results as the acemannan yield increases several fold in the in vitro regenerated callus cultures and Aloe barbadensis plants. The formation of acemannan in cultured cells was induced by mannose and sucrose carbohydrates added to the culture media.
The identity of acemannan in Aloe vera gel preparations can be significantly assessed by ¹H-NMR and FT-IR spectroscopy. Acemannan is a linear polysaccharide composed by β-(1→4)-linked mannan partially acetylated in positions 2, 3 or 6. These
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acetyl groups generate a characteristics signal (2.0-2.3 ppm) in ¹H-NMR, which shows the characteristic presence of acemannan and is considered as the fingerprint of acemannan in Aloe vera. Similarly, the results obtained by the ¹H-NMR and FT-IR spectrum of Aloe vera callus and gel extracts confirm the presence of the immunomodulatory polysaccharide acemannan.
HPLC analysis also provides strong evidence for the qualitative and quantitative analysis of bioactive compounds. The extracts of callus cultures subjected to different carbohydrate stress treatments and kept under dark conditions for 30 days were analyzed for acemannan content by HPLC. Of the different sugars and their concentrations tested, mannose and sucrose were found to be prominent in enhancing the production of this biologically potent polysaccharide. Callus cultures incubated in culture media having 3% mannose sugar produced the highest concentration of acemannan i.e 0.95 mg/mL, which revealed a remarkable enhancement of acemannan production as compared to the in vivo propagation of
Aloe vera plant in soil conditions giving yield of 0.43 mg/mL.
Calli cultured in media with 3% sucrose only increased 2% production of this compound in vitro giving 0.44 mg/mL yield. On the contrary, when glucose and galactose were added in the culture media the yield of acemannan was suppressed in all the tested concentrations.
Evaluation of Genetic Stability/ Variability of Aloe vera plants by RAPD and ISSR
Marker Assay: In the present study Random Amplified Polymorphic DNA (RAPD) and
Inter Simple Sequence Repeats (ISSR) marker assays were employed to investigate polymorphism level of conventionally grown wild type and in vitro propagated Aloe
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vera plants. Despite having phenotypic similarities in the plantlets, variation in the genomic constituents has been effectively established through RAPD and ISSR markers showing 33 % and 25% polymorphism respectively.
Biotransformation of adrenosterone: Biotransformation studies on cell suspension culture of Aloe barbadensis showed three types of reactions when compound 1 was incubated with cell suspension culture of A.barbadensis included the formation of C=C moiety at C-1/C-2 and yielded 1-2-dehydroadrenosterone (2), reduction of C-4/C-5 olefinic double bond resulted the formation of 5-androst-1-ene-3,11,17-trione (3) and the reduction of C-17 keto group, which gave 17-hydroxyandrost-4-ene-3,11-dione
(4). Structures of transformed metabolites were elucidated through comparison of their reported data. Compounds 2 and 4 were previously reported as metabolites of 1, with various fungal biotransformations.
O 18 O 12 17 16 19 11 13 H 14 15
1 9 10 8 2 H H 3 5 4 6 7 O 1
O O OH O O O
H H H H H H
O O O H 4 2 3
Scheme 1: Biotransformation of compound 1 by Cell suspension culture of Aloe barbadensis
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In conclusion, with the techniques used currently we are able to successfully improve the production of this medicinally potent compound Aloe mannan. However, complete understanding of acemannan biosynthesis and enhancement on genetic level is a complex process and requires years of research and development. These findings may play a vital role in the improvement of the natural drug based pharmaceutical industry.
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ABSTRACT (Urdu)
ﺧﻼﺻہ
ﭘﻮدے ﻗﺪﻳﻢ دور ﺳﮯ ادوﻳﺎت ﮐﮯ ﺣﺼﻮل ﮐﺎ اﻳﮏ ﻣﺜﺎﻟﯽ ذرﻳﻌہ ﮨﻴﮟ- ﺁج ﭘﻮدوں ﺳﮯ ﺣﺎﺻﻞ ﮐﺮدﮦ وﺳﻴﻊ ﺗﺮ ﻓﻌﺎل اﺻﻮل ﻣﺘﻌﺪد ﮐﻴﻤﻴﺎﺋﯽ ادوﻳﺎت ﮐﯽ ﻧﻤﺎﺋﻨﺪﮔﯽ ﮐﺮﺗﮯ ﮨﻴﮟ۔ ﺗﻘﺮﻳﺒﺎ 40 ﻓﻴﺼﺪ ﻣﺮﮐﺒﺎت ﺟﻮ دواﺳﺎزﯼ ﮐﯽ ﺻﻨﻌﺖ ﻣﻴﮟ اﺳﺘﻌﻤﺎل ﮨﻮ ر ﮨﮯ ﮨﻴﮟ ﺑﺮاﮦ راﺳﺖ ﻳﺎ ﺑﺎﻟﻮاﺳﻄہ ﭘﻮدوں ﺳﮯ ﺣﺎﺻﻞ ﮐﻴﮯ ﺟﺎﺗﮯ ﮨﻴﮟ ﮐﻴﻮﻧﮑہ اس ﻃﺮح ﮐﮯ ﻣﺮﮐﺒﺎت ﮐﯽ ﮐﻴﻤﻴﺎﺋﯽ ﺗﺮﮐﻴﺐ اﻗﺘﺼﺎدﯼ ﻃﻮر ﭘﺮ ﻳﺎ ﺗﻮ ﻣﻤﮑﻦ ﻧﮩﻴﮟ ﮨﮯ ﻳﺎ ﻗﺎﺑﻞ ﻋﻤﻞ ﻧﮩﻴﮟ ﮨﮯ۔ ﭘﻼﻧﭧ Subtropical اور (Tropical) اﺷﻨﮑﭩﺒﻨﺪﻳﯽ اﻳﮏ ﺑﮩﺖ زﻳﺎدﮦ ﻋﻼج ﮐﯽ ﺻﻼﺣﻴﺖ رﮐﻬﻨﮯ واﻻ(Aloe barbadensis)اﻳﻠﻮ وﻳﺮا ﮨﮯ۔ اﻳﻠﻮ ﺗﺎزﮦ ﺟﻴﻞ، رس ﻳﺎ ﺗﻴﺎر ﻣﺼﻨﻮﻋﺎت ﺻﺪﻳﻮں ﺳﮯ دواؤں اور ﮐﺎﺳﻤﻴﭩﮏ ﻣﻘﺎﺻﺪ ﮐﮯ ﻟﺌﮯ اﺳﺘﻌﻤﺎل ﮨﻮ ر ﮨﮯ ﮨﻴﮟ۔ Accetylated Mannan ﺟﻮ Aloe Mannan ﮐﮯ ﻃﻮر ﭘﺮ ﺑﻬﯽ ﺟﺎﻧﺎ ﺟﺎﺗﺎ ﮨﮯ ﭘﺘﯽ ﮐﮯ وا ﺿﺢ اﻧﺪروﻧﯽ ﺣﺼﮯ ﻣﻴﮟ ﮔﻮدے ﮐﮯ اﻧﺪر اﻳﮏ ﺳﭩﻮرﻳﺞ Polysaccharide ﮨﮯ۔ اس ﻣﻨﻔﺮد bioactive
ﮐﻤﭙﺎؤﻧﮉ Aloe Mannan ﮐﯽ ﻋﻠﻴﺤﺪﮔﯽ ﻧﮯ ﺑﮩﺖ ﺳﮯ ﺗﺤﻘﻴﻖ ﮐﺎروں ﮐﻮ اﭘﻨﯽ ﻃﺮف ﻣﺘﻮﺟہ ﮐﻴﺎ ﮨﮯ۔ Aloe Mannan اور اﻳﻠﻮ وﻳﺮا ﮐﯽ دﻳﮕﺮ ﻃﺒﯽ ﻟﺤﺎظ ﺳﮯ ﻓﺎﺋﺪﮦ ﻣﻨﺪﻣﺼﻨﻮﻋﺎت ﮐﯽ ﮐﻴﻤﻴﺎﺋﯽ ﺗﺮﮐﻴﺐ ﮐﺎ ﻋﻤﻞ ﻣﺸﮑﻞ اور ﻣﮩﻨﮕﺎ ﮨﮯ۔ ﭘﻼﻧﭧ ﭨﺸﻮ ﮐﻠﭽﺮ اﻳﮏ اﻣﻴﺪاﻓﺰا ﭨﻴﮑﻨﺎﻟﻮﺟﯽ ﮨﮯ، ﺧﺎص ﻃﻮر ﭘﺮ ﻧﺌﮯ اور ﺑﮩﺘﺮ ﭘﻮدوں ﮐﯽ ﺗﻴﺰرﻓﺘﺎر ﻧﺸﻮﻧﻤﺎ اور ﭘﻴﺪاوار ﮐﮯ ﻟﺌﮯ اور ﺻﻨﻌﺘﯽ اور ﻃﺒﯽ اﮨﻤﻴﺖ ﮐﯽ ﺣﺎﻣﻞ ﻣﺼﻨﻮﻋﺎت ﮐﯽ
ﺑﮍهﺘﯽ ﮨﻮﺋﯽ ﺣﻴﺎﺗﯽ ﺗﺎﻟﻴﻒ (biosynthesis) ﮐﮯ ﻟﺌﮯ۔
Micropropagation ﻣﻄﺎﻟﻌہ ﻣﻮﺟﻮدﮦ ﻣﻄﺎﻟﻌہ ﻣﻴﮟ اﻳﮏ اﻋﻠﯽ Aloe barbadensisﮐﯽ ﻗﺴﻢ ﮐﻮ ، acemannan ﮐﯽ اوﻧﭽﯽ ﺳﻄﺢ ﮨﻮﻧﮯ ﮐﯽ وﺟہ ﺳﮯ ﻣﻨﺘﺨﺐ ﮐﻴﺎ ﮔﻴﺎ ﺗﻬﺎ اور اﻳﮏ ﻣﻤﮑﻨہ ﺑﻼواﺳﻂ ﺗﺨﻠﻴﻖ ِﻧﻮ ﮐﯽ ﭘﻴﺮوﯼ ﻣﻴﮟ ﮔﻬﭩﭩﺎ (Callus)ﮐﯽ اﺑﺘﺪائ ﻧﺸﻮﻧﻤﺎ ﮐﯽ ﺗﺤﻘﻴﻖ ﮐﯽ ﮔﺊ ۔ روﺷﻨﯽ ﮐﮯ ﻣﻘﺎﺑﻠﮯ ﻣﻴﮟ زﻳﺎدﮦ ﺳﮯ زﻳﺎدﮦ calli ﮐﻮ ﺗﺎرﻳﮑﯽ ﻣﻴﮟ Murashige) MS اور Skoog) "ﻃﺮﻳﻘﮯ" ﭘﺮ ﺗﺮﻏﻴﺐ دﯼ ﮔﺊ mg/L of 2-8
(NAA (α-naphthaleneacetic acidﮐﮯ اﺿﺎﻓہ ﮐﮯ ﺳﺎﺗﻪ۔ اس ﮐﮯ ﺑﺮﻋﮑﺲ، ﺷﺎﺧﻮں ﮐﯽ ﺗﺨﻠﻴﻖ ﻧﻮ
ﺳﺐ ﺳﮯ زﻳﺎدﮦ ﻓﺮﻳﮑﻮﺋﻨﺴﯽ ﭘﺮ calli ﮐﯽ ذﻳﻠﯽ ﮐﻠﭽﺮﻳﻨﮓ ﮐﮯ ذرﻳﻌہ,MS mediumﻣﺸﺘﻤﻞ mg/L 0.1 (of IBA (Indole-3-butyric acidاور(mg/L of BAP (6-Benzyl amino purine 1.0 ﭘﺮتlight ﮐﮯ ﻣﺎﺣﻮل ﻣﻴﮟ ﺣﺎﺻﻞ ﮐﯽ ﮔﺊ۔ ﭘﻮدوں ﻧﮯ ﺗﺨﻠﻴﻖ ﻧﻮﮐﮯ ﻳﮑﺴﺎں medium ﭘﺮ اﭼﻬﯽ
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ﻃﺮح ﺟﮍﻳﮟ ﭘﮑﮍﻳﮟ اور ﮔﺮﻳﻦ ﮨﺎؤس ﮐﮯ ﺣﺎﻻت ﺳﮯ ﮐﺎﻣﻴﺎﺑﯽ ﺳﮯ ﻣﺎﻧﻮس ﮨﻮے۔ ﺗﺠﺮﺑﺎت ﮐﮯ ﺳﻠﺴﻠﮯ ﻣﻴﮟ ﻣﻤﮑﻨہ in vitroﺟﻨﻴﺎﺗﯽ ﺗﺒﺪﻳﻠﯽ ﮐﯽ ﺗﺤﻘﻴﻘﺎت ﮐﺮﻧﮯ ﮐﯽ ﻏﺮض ﺳﮯ ﮔﻬﭩﭩﺎ (calli) ﮐﻠﭽﺮ ﻣﺨﺘﻠﻒ دﺑﺎؤ ﮐﮯ ﻣﺎﺣﻮل ﺳﮯ ﮔﺰارے ﮔﺌﮯ ﺟﺲ ﮐﮯ ﻧﺘﻴﺠہ ﻣﻴﮟ ﮔﻬﭩﭩﺎ (calli) اور in vitro ﮐﻠﻴﺲ(callus) ﭨﺸﻮ ﺳﮯ ﺣﺎﺻﻞ ﮐﺮدﮦ ﺗ ﺨ ﻠ ﻴ ﻖِ ﻧﻮ ﭘﻮدوں ﻣﻴﮟ" immunomodulatory polysaccharide acemannan" ﮐﯽ اﻓﺰودﮦ ﭘﻴﺪاوار ﺣﺎﺻﻞ ﮨﻮﯼ۔ اﻳﻠﻮ وﻳﺮا calli ﮐﻮ ﭼﺎر ﻣﺨﺘﻠﻒ ﮐﺎرﺑﻦ ذراﺋﻊ (glucose ،mannose ، sucrose اور galactose) اور ﭘﺎﻧﭻ concentrations (2، 3، 4، 5 اور٪6) ﮐﮯ ﺳﺎﺗﻪ in vitro ﮐﻠﭽﺮ ﮐﻴﺎ ﮔﻴﺎ اور 1 ﻣﺎﮦ ﺗﮏ ﺗﺎرﻳﮑﯽ ﻣﻴﮟ رﮐﻬﺎ ﮔﻴﺎ۔ ﭘﻬﺮ ﮐﻠﭽﺮز ﮐﺎ ﺗﺠﺰﻳہ ﮐﻴﺎ ﮔﻴﺎ acemannan ﻣﻮاد ﺟﺎﻧﭽﻨﮯ ﮐﮯﻟﻴﮯ ﺟﻮ ﮐﮯ mannose 3٪ ﭘﺮﻣﺸﺘﻤﻞ ﺷﻮﮔﺮ mediumﻣﻴﮟ ﺳﺐ ﺳﮯ زﻳﺎدﮦ ﭘﺎﻳﺎ ﮔﻴﺎ۔ اس ﻃﺮح ﺳﮯ ﺣﺎﺻﻞ ﮐﺮدﮦ ﻧﺘﺎﺋﺞ ﮐﮯ ﻣﻄﺎﺑﻖ ﻳہ واﺿﺢ ﺗﻬﺎ ﮐہ bioactive“ ”polysaccharide acemannanﮐﯽ ﭘﻴﺪاوار in vitro ﺗ ﺨ ﻠ ﻴ ﻖِ ﻧﻮ اﻳﻠﻮ وﻳﺮا ﭘﻼﻧﭩﺲ ﻣﻴﮟ اﺿﺎﻓﯽ ﺗﻬﯽ، رواﻳﺘﯽ ﻃﺮﻳﻘﮯ ﺳﮯ ﮐﻬﻴﺖ ﻣﻴﮟ ﻟﮕﺎے ﮔﺌﮯ ﭘﻮدوں ﮐﮯﻣﻘﺎﺑﻠﮯ ﻣﻴﮟ۔ اس ﻃﺒﯽ ﮐﻤﭙﺎؤﻧﮉ ﮐﮯ ﻣﻘﺪارﯼ اور ﮐﻴﻔﻴﺎﺗﯽ ﺗﺠﺰﻳہ ﮐﮯ ﻟﺌﮯ اﻧﻔﺮارﻳﮉﺳﭙﻴﮑﭩﺮوﺳﮑﻮﭘﯽ، 1H-NMR اور HPLC ﮐﯽ ﺗﮑﻨﻴﮏ اﺳِﺘﻤﺎل ﮐﯽ ﮔﺊ۔
Acemannan اﻓﺰوﻧﯽ: "Bioactive Polysaccharide Acemannan" ﮐﯽ ﺑﮩﺘﺮ ﭘﻴﺪاوار ﮐﮯ ﻟﺌﮯ in vitro ﭘﻮدوں ﭘﺮ ﺗﺤﻘﻴﻖ ﮐﮯ ﻟﺌﮯ ﮐﻴﻠﺲ ﮐﮯ راﺳﺘﮯ ﮐﻮ اﭘﻨﺎﻳﺎ ﮔﻴﺎ۔ دﺑﺎو ﮐﯽ ﮐﻴﻔﻴﺖ ﻣﻴﮟ ﭘﻴﺪاوار ﮐﮯ ذراﺋﻊ اور روﺷﻨﯽ ﮐﯽ ﺻﻮرﺗﺤﺎل ﻧﮯ acemannan ﮐﯽ ﺳﻄﺢ ﻣﻴﮟ اﺿﺎﻓہ ﮐﺮﻧﮯ ﻣﻴﮟ اﻳﮏ اﮨﻢ ﮐﺮدار ادا ﮐﻴﺎ ۔ ﺗﺤﻘﻴﻘﺎت ﺳﮯ ﻧﮩﺎﻳﺖ دﻟﭽﺴﭗ ﻧﺘﺎﺋﺞ ﺳﺎﻣﻨﮯ ﺁﺋﮯ ﺟﻴﺴﮯ ﮐہ in vitro ﮐﻠﻴﺲ (callus) ﮐﻠﭽﺮ ﮐﯽ ﻧﻤﻮدِﻧﻮ اور اﻳﻠﻮ barbadensis ﭘﻮدوں ﻣﻴﮟ acemannan ﮐﯽ ﭘﻴﺪاوار ﻣﻴﮟ ﮐﺌﯽ ﮔﻨﺎ اﺿﺎﻓہ دﻳﮑﻬﺎ ﮔﻴﺎ۔ ﮐﻠﭽﺮڈ ﺧﻠﻴﺎت ﻣﻴﮟ acemannan ﮐﯽ ﺗﺸﮑﻴﻞ ﮐﻠﭽﺮ ﻣﻴﮉﻳﺎ ﻣﻴﮟ mannose اور sucrose ﮐﺎرﺑﻮﮨﺎﺋﻴﮉرﻳﭧ ﺷﺎﻣﻞ ﮐﺮﻧﮯ ﮐﯽ ﻣﺮﮨﻮن ِﻣﻨﺖ ﮨﮯ۔ اﻳﻠﻮ وﻳﺮا ﺟﻴﻞ ﮐﯽ ﺗﻴﺎرﯼ ﻣﻴﮟ acemannan ﮐﯽ ﺷﻨﺎﺧﺖ ﻧﻤﺎﻳﺎں ﻃﻮر ﭘﺮ1H-NMR اور FT-IR ﮐﮯ ذرﻳﻌﮯ ﮐﯽ ﺟﺎﺳﮑﺘﯽ ﮨﮯ۔ Acemannan ﺳﻴﺪهﯽ ﻟﮑﻴﺮ ﭘﺮﻣﺸﺘﻤﻞ اﻳﮏ polysaccharide ﮨﮯ ﺟﻮ (β-(1→4 ﺳﮯﻣﺮﺗﺐ ﮨﮯ اور mannanﮐﻮ ﺟﺰوﯼ ﻃﻮر ﭘﺮ 2، 3 ﻳﺎ 6 ﺳﮯﺗﻌﻠﻖ رﮐﻬﻨﮯ واﻟﯽ ﭘﻮزﻳﺸﻦ ﺳﮯ ﺟﻮڑﺗﺎ ﮨﮯ۔ ﻳہ acetyl ﮔﺮوپ ¹H-NMR ﻣﻴﮟ (ppm 2.0-2.3) ﮐﺎ ﺧﺼﻮﺻﯽ اﺷﺎرا دﻳﺘﮯ ﮨﻴﮟ ﺟﺲ ﺳﮯ acemannan ﮐﯽ ﺧﺼﻮﺻﻴﺎت ﮐﯽ ﻣﻮﺟﻮدﮔﯽ ﮐﺎ ﭘﺘہ ﭼﻠﺘﺎ ﮨﮯ اوراﻳﻠﻮ وﻳﺮا ﻣﻴﮟ acemannan ﮐﮯ ﻓﻨﮕﺮ ﭘﺮﻧﭧ ﮐﮯ ﻃﻮر ﭘﺮ ﺳﻤﺠﻬﺎ ﺟﺎﺗﺎ ﮨﮯ۔ اﺳﯽ ﻃﺮح، -1H NMR اور FT-IR ﺳﭙﻴﮑﭩﺮم ﮐﮯ اﻳﻠﻮ وﻳﺮا ﮐﻴﻠﺲ اور ﺟﻴﻞ ﻋﺮق ﮐﮯ ﺑﺎرے ﻣﻴﮟ ﺣﺎﺻﻞ ﺷﺪﮦ ﻧﺘﺎﺋﺞ immunomodulatory polysaccharide acemannan ﮐﯽ ﻣﻮﺟﻮدﮔﯽ ﮐﯽ ﺗﺼﺪﻳﻖ ﮐﺮﺗﮯ ﮨﻴﮟ۔
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HPLC ﺗﺠﺰﻳہ ﺑﻬﯽ bioactive ﻣﺮﮐﺒﺎت ﮐﮯ ﺻﻔﺎﺗﯽ اور ﻣﻘﺪارﯼ ﺗﺠﺰﻳہ ﮐﮯ ﻟﺌﮯ ﻣﻀﺒﻮط ﺛﺒﻮت ﻓﺮاﮨﻢ ﮐﺮﺗﺎ ﮨﮯ۔ ﮐﻠﻴﺲ(Callus) ﮐﻠﭽﺮ ﮐﮯ ﻋﺮق ﮐﻮ 30 دﻧﻮں ﮐﮯ ﻟﺌﮯ ﻣﺨﺘﻠﻒ ﮐﺎرﺑﻮﮨﺎﺋﮉرﻳﭧ دﺑﺎو ﮐﮯ زﻳﺮﻳﮟ ﺗﺎرﻳﮑﯽ ﻣﻴﮟ رﮐﻬﺎ ﮔﻴﺎ اور HPLCﮐﮯ زرﻳﻌہ ﺳﮯ acemannan ﻣﻮاد ﮐﮯ ﻟﺌﮯ ﺗﺠﺰﻳہ ﮐﻴﺎ ﮔﻴﺎ۔ ﻣﺨﺘﻠﻒ اﻗﺴﺎم ﮐﯽ ﺷﮑﺮ اور ان ﮐﯽ ﺗﻌﺪاد ﺟﻦ ﮐﺎ ﺟﺎﺋﺰﮦ ﻟﻴﺎ ﮔﻴﺎ، ان ﻣﻴﮟ mannoseاور sucrose اس ﺣﻴﺎﺗﻴﺎﺗﯽ ﻗﻮﯼ polysaccharideﮐﯽ ﭘﻴﺪاوار ﺑﮍهﺎﻧﮯ ﻣﻴﮟ ﻧﻤﺎﻳﺎں ﭘﺎﺋﮯ ﮔﺌﮯ۔ ﮐﻠﻴﺲ(Callus) ﮐﻠﭽﺮﺟﻮ ﮐہ mannose 3٪ ﺷﮑﺮ ﮐﮯ ﮐﻠﭽﺮ ﻣﻴﮉﻳﺎ ﻣﻴﮟ incubation ﮐﮯﻋﻤﻞ ﺳﮯ ﮔﺰارے ﮔﺌﮯ acemannan ﮐﯽ ﺳﺐ ﺳﮯ زﻳﺎدﮦ ﺗﻌﺪاد ﭘﻴﺪا ﮐﺮﻧﮯ ﮐﺎ ﺑﺎﻋﺚ ﺛﺎﺑﺖ ﮨﻮےﻳﻌﻨﯽ ﮐہ mg/mL 0.95 ﺟﺲ ﺳﮯ acemannan ﮐﯽ ﭘﻴﺪاوار ﻣﻴﮟ اﻳﮏ ﻗﺎﺑﻞ ذﮐﺮ اﺿﺎف ﮐﺎ اﻧﮑﺸﺎف ﮨﻮﺗﺎ ﮨﮯ ، اﻳﻠﻮ وﻳﺮا ﮐﮯ ﭘﻼﻧﭧ ﮐﯽ زﻣﻴﻦ ﻣﻴﮟ in vitro ﺗﺮوﻳﺞ ﮐﮯ ﻣﻘﺎﺑﻠﮯ ﻣﻴﮟ ﺟﺲ ﮐﯽ ﭘﻴﺪاوار mg/mL 0.43 ﺗﻬﯽ۔ Calliﺟﻮ ﮐﮯ sucrose 3٪ ﻣﻴﮉﻳﺎ ﻣﻴﮟ ﮐﻠﭽﺮڈ ﮐﻴﮯ ﮔﺌﮯ ﺗﻬﮯ اس ﮐﻤﭙﺎؤﻧﮉ in vitro ﮐﯽ ﭘﻴﺪاوار ﻣﻴﮟ ﺻﺮف٪2 اﺿﺎﻓہ ﮐﺎ ﺳﺒﺐ ﺑﻨﮯ ﻳﻌﻨﯽ mg/mL 0.44 اس ﮐﮯ ﺑﺮﻋﮑﺲ، ﺟﺐ ﮔﻠﻮﮐﻮز اور galactose ﮐﻮﮐﻠﭽﺮ ﻣﻴﮉﻳﺎ ﻣﻴﮟ ﺷﺎﻣﻞ ﮐﻴﺎ ﮔﻴﺎ ﺗﻮ acemannan ﮐﯽ ﭘﻴﺪاوار ﺗﻤﺎم ﺗﺠﺮﺑﺎت ﻣﻴﮟ دب ﮔﺌﯽ۔
RAPD اور ISSR ﻣﺎرﮐﺮ ﺟﺎﻧﭻ ﮐﮯ زرﻳﻌﮯ ﺳﮯ اﻳﻠﻮ وﻳﺮا ﭘﻮدوں ﮐﺎ ﺟﻴﻨﻴﺎﺗﯽ اﺳﺘﺤﮑﺎم / ﺗﻐﻴﺮ ﭘﺬﻳﺮﯼ ﮐﺎ اﻧﺪازﮦ : ﻣﻮﺟﻮدﮦ ﻣﻄﺎﻟﻌہ ﻣﻴﮟ RAPD) Random Amplified Polymorphic DNA) اور اﻧﭩﺮ ﺳِﻤﭙﻞ ﺗﺴﻠﺴﻞ ﺗﮑﺮار (ISSR) ﻣﺎرﮐﺮ ﺟﺎﻧﭻ ﮐﮯ زرﻳﻌﮯ ﺟﻨﮕﻠﯽ ﻗﺴﻢ ﮐﯽ رواﻳﺘﯽ ﭘﻴﺪاوار اور in vitro اﻓﺰاﺋﺸﯽ اﻳﻠﻮ وﻳﺮا ﭘﻮدوں ﻣﻴﮟ ﺑﮩﺮوﭘﺘﺎ (Polymorphism) ﮐﯽ ﺳﻄﺢ ﮐﯽ ﺗﺤﻘﻴﻘﺎت ﮐﯽ ﮔﺌﻴﮟ۔ ﭼﻬﻮﭨﮯ ﭘﻮدوں ﻣﻴﮟ ﺷﮑﻠﯽ phenotypic ﻣﻤﺎﺛﻠﺖ ﮨﻮﻧﮯ ﮐﮯ ﺑﺎوﺟﻮد، ﺟﻴﻨﻮﻣﮏ اﺟﺰاء ﻣﻴﮟ ﺗﺒﺪﻳﻠﯽ ﮐﻮ ﻣﺆﺛﺮ ﻃﺮﻳﻘﮯ ﺳﮯ ﺑﺎﻟﺘﺮﺗﻴﺐ٪33 اور ٪25 ﺑﮩﺮوﭘﺘﺎ (Polymorphism) دﮐﻬﺎﺗﮯ ﮨﻮﺋﮯ RAPD اور ISSR ﻣﺎرﮐﺮ ﮐﮯ ذرﻳﻌﮯ ﻗﺎﺋﻢ ﮐﻴﺎ ﮔﻴﺎ ﮨﮯ-
Adrenosterone ﮐﯽ ﺣﻴﺎﺗﯽ ﺗﺒﺪﻳﻠﯽ: ﺣﻴﺎﺗﯽ ﺗﺒﺪﻳﻠﯽ Biotransformation ﮐﮯ ﻣﻄﺎﻟﻌہ ﻧﮯ ﻇﺎﮨﺮ ﮐﻴﺎ ﮐہ barbadensis Aloe ﮐﮯ ﺳﻴﻞ ﺳﺴﭙﻴﻨﺸﻦ ﮐﻠﭽﺮﻧﮯ ﺗﻴﮟ ﻗﺴﻢ ﮐﮯ ردِﻋﻤﻞ ﮐﺎ ﻣﻈﺎﮨﺮﮦ ﮐﻴﺎ ﺟﺐ ﮐﻤﭙﺎؤﻧﮉ 1 ﮐﻮ Aloe barbadensis ﮐﮯ ﺳﻴﻞ ﺳﺴﭙﻴﻨﺸﻦ ﮐﻠﭽﺮ ﮐﮯ ﺳﺎﺗﻪ incubate ﮐﻴﺎ ﮔﻴﺎ ﺑﺸﻤﻮل C-1/C-2 ﻣﻴﮟ C = C moiety ﮐﯽ ﺗﺸﮑﻴﻞ 1-2 ﻣﻴﮟ اور -dehydroadrenosterone ﺣﺎﺻﻞ ﮨﻮا۔ (C-4/C-5 olefinic (2 ڈﺑﻞ ﺑﺎﻧﮉ ﮐﯽ ﮐﻤﯽ ﮐﺎ
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ﻧﺘﻴﺠہ,trione 5-androst-1-ene-3,11 -17 ﺑﺮاﻣﺪ ﮨﻮا (3) او C-17 keto ﮔﺮوپ ﮐﯽ ﮐﻤﯽ ﺳﮯ 17-hydroxyandrost-4-ene-3,11-dione ﺣﺎﺻﻞ ﮨﻮا۔ (4) ﺗﺒﺪﻳﻞ ﺷﺪﮦ metabolites ﮐﮯ ڈهﺎﻧﭽﻮں ﮐﺎ ﺗﻌﻴﻦ ان ﮐﮯ ﭘﻴﺶ ﮐﺮدﮦ اﻋﺪاد و ﺷﻤﺎر ﺳﮯ ﻣﻮازﻧہ ﮐﮯ ذرﻳﻌﮯ ﮐﻴﺎ ﮔﻴﺎ۔ ﻣﺮﮐﺒﺎت 2 اور ﮐﻮ ﭘﮩﻠﮯ ﻣﺨﺘﻠﻒ ﭘﻬﭙﻬﻮﻧﺪﯼ(fungal) ﮐﯽ ﺣﻴﺎﺗﯽ ﺗﺒﺪﻟﯽ ﮐﮯ ﺳﺎﺗﻪ، 1 ﮐﮯ metabolites ﮐﮯ ﻃﻮر ﭘﺮﭘﻴﺶ ﮐﻴﺎ ﮔﻴﺎﺗﻬﺎ۔
O 18 O 12 17 16 19 11 13 H 14 15
1 9 10 8 2 H H 3 5 4 6 7 O 1
O O OH O O O
H H H H H H
O O O H 4 2 3
ﺳﮑﻴﻢ Aloe Barbadensis :1 ﮐﮯ ﺳﻴﻞ ﻣﻌﻄﻠﯽ culture ﮐﯽ ﻃﺮف ﺳﮯ ﮐﻤﭙﺎؤﻧﮉ 1 ﮐﯽ Biotransformation
ﻣ ﺨ ﺘ ﺼ ﺮ اً ، ﻣﻮﺟﻮدﮦ اﺳﺘﻌﻤﺎل ﮐﯽ ﺟﺎﻧﮯ واﻟﯽ ﺗﺮاﮐﻴﺐ (techniques) ﮐﮯ زرﻳﻌﮯ ﮨﻢ ﮐﺎﻣﻴﺎﺑﯽ ﺳﮯ اس ﻃﺎﻗﺘﻮر ﻃﺒﯽ ﮐﻤﭙﺎؤﻧﮉ اﻳﻠﻮ mannan ﮐﯽ ﭘﻴﺪاوار ﮐﻮ ﺑﮩﺘﺮ ﺑﻨﺎﻧﮯ ﮐﮯ ﻗﺎﺑﻞ ﮨﻴﮟ۔ ﺗﺎﮨﻢ، ﺟﻴﻨﻴﺎﺗﯽ ﺳﻄﺢ ﭘﺮ acemannan ﺣﻴﺎﺗﯽ ﺗﺎﻟﻴﻒ اور اﻓﺰاﺋﺶ ﮐﯽ ﻣﮑﻤﻞ ﺗﻔﮩﻴﻢ اﻳﮏ ﭘﻴﭽﻴﺪﮦ ﻋﻤﻞ ﮨﮯ اور ﺗﺤﻘﻴﻖ اور ﺁزﻣﺎﺋﺶ ﮐﮯﮐﺌﯽ ﺳﺎﻟﻮں ﮐﯽ ﺿﺮورت ﮨﮯ۔ ﻳہ ﻧﺘﺎﺋﺞ ﻗﺪرﺗﯽ ادوﻳﺎت ﮐﯽ ﺑﻨﻴﺎد ﭘﺮ دوا ﺳﺎزﯼ ﮐﯽ ﺻﻨﻌﺖ ﮐﯽ ﺑﮩﺘﺮﯼ ﻣﻴﮟ اﮨﻢ ﮐﺮدار ادا ﮐﺮ ﺳﮑﺘﮯ ﮨﻴﮟ.
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ACKNOWLEDGEMENTS
“And for those who fear Allah, He (ever) prepares a way out,
And He provides for him from (sources) he never could imagine.
And if anyone puts his trust in Allah, sufficient is Allah for him. For
Allah will surely accomplish his purpose; verily, for all things has
Allah appointed a due proportion”.
Sura At‐ Talaq Ayat nos 2 (part) and 3
It gives me a great pleasure to express my gratitude and profound veneration to all those people who have extended their support and cooperation during the entire process of my Ph.D. research work and enabled me to accomplish my greatest achievement.
First and foremost, I am extremely thankful to Almighty Allah (The most Gracious and
Merciful), who has always guided and protected me and given me strength and courage to overcome the challenges in life. May Peace and Blessing of Allah be upon
His Prophet Muhammad (P.B.U.H.).
I would like to express my deepest gratitude to my respected supervisor and mentor,
Dr. Saifullah Khan, Associate Professor and In-charge Biotechnology Wing, H.E.J.
Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, for believing in me and providing me the opportunity to work under his esteemed supervision. His innovative teaching, immense support, patience and constant guidance were the key motivations in the course of development and completion of this doctoral study.
My sincere acknowledgements and profound regards to Dr. Mustafa Kamal, Associate
Professor and In-charge Department of Biotechnology, University of Karachi and to all
xiii
my teachers for their devoted instruction and enlightening guidance that proved to be a stepping stone for developing my aptitude for scientific research.
My cordial gratitude goes to Dr. Shakil Ahmed, Dr. Syed Abid Ali, Dr. Muhammad
Shaiq Ali, Dr. Syed Ghulam Musharraf, Dr. Sammer Yousuf and Dr. Achyut Adhikari for their expertise, kind assistance and valuable suggestions.
I would like to express my sincere acknowledgement and appreciation to Prof. Dr.
Muhammad Iqbal Choudhary, Director, H.E.J. Research Institute of Chemistry,
International Center for Chemical and Biological Sciences, University of Karachi, for providing state of the art research and educational facility in Pakistan.
I am thankful to all the technical and non technical staff members of I.CC.B.S and
Biotechnology Wing; Abdul Ghaffar, Khalid Hussain, M. Tahir, Qurban Ali, Tariq Rao and Zafar Iqbal, for their help, guidance and cooperation in facilitating my research work.
It is my pleasure to acknowledge all my current and previous colleagues and friends especially Asma Nasib, Dr. Bushra Noman, Naheed Kauser, Sheeba Naz, Dr. Mariam
Raziq, Kahkashan Kazmi, Huma Rao, Darakshan Imran and Noor Afsah for their helpful discussions, enormous support and cooperation. Without them this project would not have been completed. I am grateful to Dr. Saifullah Sahir, Dr. Ameer
Ahmed, and Hammad Afzal Kayani for all their help and critical review of my thesis.
I fall short of words and can’t imagine my current position without the unconditional love, constant support and encouragement of my family which is the true essence of my life. My special thanks goes to my dearest loving husband and lovely kids, M.
Faizan, Ayesha and M. Nabeel, who have endured a lot during the entire process.
They have always extended their total cooperation and allowed me to dedicate full time to this project with complete concentration and focus. I would like to pay special tribute to my husband Muhammad Badar Arshi, who always stood by my side through thick and thin and has been a great source of inspiration and motivation. I am deeply
xiv
obliged for all his sacrifices, efforts and support he has rendered for completing my
Ph.D, which kept me optimistic, even during the most difficult times.
My heartiest appreciations and gratitude to my dearest loving brother Nasir Iftikhar, sister-in-law Ana Hita Temuri, my lovely niece and nephews and specially my dearest
Aunty Nighat Sultana, who have always been there for me in time of need. This journey would not have been possible without all of you. Thank you for everything.
I am especially thankful to my Uncle M. Saleem Siddique and his family for bringing the acemannan standard from U.S.A.
I am highly grateful and indebted to my in-laws for their prayers, kindness, sincere cooperation and encouragement which facilitated my studies. My heartfelt appreciation to my father-in-law M. Irshad Zaffar and mother-in-law Surryia Kauser for taking care of my home and kids in my absence, despite their illness and limitations.
My sincere acknowledgement goes to the families of my sisters-in-law Ambreen
Noman, Nadia Nadeem and brothers-in-law for their immense support and constant motivation. Very special thanks to M. Faheem Irshad and Zeeshan Ali Khan for always extending their brotherly help.
Finally, my highest regard goes to my extremely loving and caring parents
Iftikharuddin Sheikh and Roohi Iftikhar who always pray for my success and praise my achievements. Their sacrifices, continuous guidance, endless support and encouragement have made every opportunity available to me throughout my life.
Thank You
Zarreen Badar
xv List of Abbreviations
ABBREVIATIONS
ABA Abscisic acid A Adenine CAN Acetonitrile ANOVA Analysis of variance B Boron BAP Benzylaminopurine
bp Base pair
C Cytosine 0C Degree Celsius Ca Calcium CA ` Co autoclavable
Ca(ClO)2. Calcium hypochlorite
Ca[NO3]2.4H2O Calcium nitrate
CaCl2.2H2O Calcium chloride Cl Chloride Cm Centimeter CTAB Hexadecyltrimethyl-amonium bromide
CuSO4.5H2O Cupric sulphate oC Degree Centigrade DMRT Duncan Multiple Range Test dATP 2’ deoxyadenosine 5’ triphosphate dCTP 2’ deoxycytidine 5’ triphosphate dGTP 2’ deoxyguanosine 5’ triphosphate dTTP 2’ deoxythymidine 5’ triphosphate DNA Deoxy ribose nuclic acid dNTPs Deoxyribonucleoside triphosphate EDTA Ethylenediamine tetra acetic acid (Merck,charge/Lot#K2133256) G Guanine 2,4-D Dichlorophenoxyacetic Acid Fe Iron
xv List of Abbreviations
FTIR Fourier transform Infrared spectroscopy Gm Gram HCl Hydrochloric acid HPLC High performance liquid chromatography Hr Hour IBA Indole-3-ButyriIc Acid ISSR Inter simple sequence repeats K Potassium KCl Potassium chloride
KH2PO4 Potassium phosphate Kin Kinetin
KNO3 Potassium nitrate L Liter mA Miliampere Mg Magnesium
MgCl2 Magnesium chloride Mg/L Milligram/liter
MgSO4.7H2O Magnesium sulfate Min Minute mL Milliliter M Molar mM Micro molar Mn Manganese
MnSO4.H2O Manganese sulfate Mo Molybdenum MS Media Murashige and Skoog (1962) basal media Ng Nanogram NMR Nuclear Magnetic Rresonance O.D. Optical density µg Microgram µl Microliter µm Micromoles NaCl Sodium chloride
xvi List of Abbreviations
Ng Nanogram N Nitrogen
Na2 Fe EDTA Ferric ethylene diamine tetra acetic acid
Na2MoO4.2H2O Sodium molybdate NAA α-Naphthalene acetic acid
NaH2PO4.H2O Sodium phosphate NaOH Sodium hydroxide
NH4 Ammonium
NO3 Nitrate Pm Picomole Pg Picogram PCR Polymerase chain reaction O.D Optical density % Percentage P Phosphorus PB Phosphate Buffer PBS Phosphate Buffer Saline RNA Ribonucleic acid RNase A Ribonulease A RAPD Random amplified polymorhic DNA Rpm Revolutions per minute RT Room Temperature S Sulfur 2 SO4 Sulfate UV Ultraviolet v/v volume/volume w/v weight/volume Zea Zeatin
Zn(C2H4O2N)2 Zinc chelate ZnO Zinc oxide
ZnSO4.7H2O Zinc sulfate
xvii
TABLE OF CONTENTS
DECLARATION ...... i DEDICATION ...... iii ABSTRACT (English) ...... iv ABSTRACT (Urdu) ...... ix ACKNOWLEDGEMENTS ...... xiii ABBREVIATIONS ...... xv
CHAPTER 1 – GENERAL INTRODUCTION
1.1 GENERAL INTRODUCTION ...... 8 1.1.1 Scope and Significance of Biotechnology ...... 8 1.1.1.1 Application of Biotechnology in Medicine ...... 8 1.1.1.2 Industrial Biotechnology ...... 8 1.1.1.3 Biotechnology and Environment ...... 9 1.1.1.4 Plant Biotechnology ...... 9 1.1.2 Medicinal Plants ...... 10 1.1.3 The Aloe vera Plant ...... 12 1.1.3.1 Scientific Classification ...... 12 1.1.3.2 Nomenclature ...... 12 1.1.3.3 Common or other Local, Multi‐lingual names ...... 13 1.1.3.4 Taxonomy and Etymology ...... 13 1.1.3.5 Aloe vera Botanical Description...... 14 1.1.3.6 Ecological Distribution ...... 14 1.1.4 Economic Importance ...... 15 1.1.4.1 Medicinal Plants in World Market ...... 15 1.1.4.2 Current Status of Medicinal Plant Resources in Pakistan ...... 15 1.1.4.3 Aloe Trade ...... 17 1.1.4.3.1 Current Market Scenario...... 17 1.1.4.3.2 Raw Material...... 17 1.1.4.3.3 Present Global Players ...... 17 1.1.5 Medicinal Importance ...... 18
i
1.1.5.1 Use of Aloe in Traditional Therapy ...... 18 1.1.5.2 Use in Modern Medicine ...... 19 1.1.6 Components of Aloe vera and their biological effects ...... 21 1.1.6.1 Pharmaceutical Activities of Aloe vera Components ...... 22 1.1.7 Propagation Techniques ...... 23 1.1.7.1 Cultivation Practices ...... 23 1.2 Plant Tissue Culture Technology ...... 26 1.2.1 Brief History ...... 26 1.2.2 Explant Source ...... 29 1.2.3 Nutrient Media ...... 29 1.2.4 Plant nutrients and their role in plant tissue culture ...... 30 1.2.4.1 Non‐mineral Elements ...... 31 1.2.4.2 Mineral Elements or In‐organic Supplements ...... 31 1.2.4.2.1 Macronutrients ...... 31 1.2.4.2.2 Micronutrients ...... 32 1.2.4.2.3 Organic Supplements ...... 33 1.2.4.2.3.1 Vitamins ...... 33 1.2.4.2.3.2 Amino Acids ...... 33 1.2.4.2.3.3 Carbon and energy sources ...... 34 1.2.4.2.4 Natural substances and extracts ...... 35 1.2.4.2.5 Activated charcoal ...... 35 1.2.4.2.6 Anti‐browning compounds ...... 36 1.2.4.2.7 Plant Growth Regulators ...... 37 1.2.4.2.7.1 Auxins ...... 37 1.2.4.2.7.2 Cytokinins ...... 38 1.2.4.2.7.3 Gibberellins ...... 39 1.2.4.2.7.4 Abscisic acid ...... 39 1.2.4.2.7.5 Ethylene ...... 40 1.2.5 The role of physical factors in plant tissue culture ...... 40 1.2.5.1 Light ...... 40 1.2.5.2 Temperature ...... 41 1.2.5.3 Media preparation ...... 41 1.2.5.4 Media sterilization ...... 42 1.2.5.5 pH ...... 42 1.2.5.6 Solidifying agent ...... 43
ii
1.2.6 Practical aspects of plant cell tissue and organ culture ...... 44 1.2.6.1 Callus Culture ...... 44 1.2.6.2 Organogenesis ...... 45 1.2.6.3 Cell Suspensions ...... 45 1.2.7 Applications and future prospects ...... 47 1.3 Research Objectives ...... 49
CHAPTER 2 – MICROPROPAGATION
2.1 INTRODUCTION ...... 52 2.1.1 Micropropagation of Aloe vera ...... 52 2.1.1.1 Advantages of Micropropagation over Conventional Propagation ...... 55 2.1.1.2 Disadvantages of Micropropagation ...... 56 2.2 MATERIALS AND METHODS ...... 57 2.2.1 Preparation of Plant Growth Regulators—Stock ...... 57 2.2.2 Media Preparation ...... 58 2.2.3 Isolation of Explants ...... 59 2.2.4 Cleaning of Explants ...... 59 2.2.5 Sterilization of Explants ...... 59 2.2.6 Inoculation of Explants on Culture Initiation Medium ...... 59 2.2.7 Callus Induction ...... 61 2.2.8 Organogenesis ...... 62 2.2.9 Acclimatization ...... 62 2.2.10 Statistical Analysis ...... 63 2.3 RESULTS AND DISCUSSION ...... 64 2.3.1 Callus Induction ...... 64 2.3.2 Effect of Light ...... 64 2.3.3 Effect of Hormones ...... 65 2.3.4 Organogenesis ...... 69 2.3.4.1 Shooting ...... 69 2.3.4.2 Effect of Light ...... 69 2.3.4.3 Effect of Hormones ...... 70 2.3.4.4 Rooting and Acclimatization ...... 71
iii
CHAPTER 3 – QUALITATIVE AND QUANTITATIVE ANALYSIS
3.1 INTRODUCTION ...... 76 3.1.1 Qualitative and Quantitative Analysis of Acemannan ...... 76 3.1.2 Acemannan Isolation and Purification Studies ...... 77 3.1.3 Chromatography ...... 79 3.1.4 A Brief History ...... 79 3.1.5 High Performance Liquid chromatography (HPLC) ...... 80 3.1.5.1 Types of HPLC ...... 82 3.1.5.1.1 Normal Phase or Adsorption Chromatography ...... 82 3.1.5.1.2 Reverse Phase Liquid Chromatography (RPLC) ...... 82 3.1.5.1.3 Ion Exchange Chromatography ...... 83 3.1.5.1.4 Size Exclusion Chromatography (SEC) ...... 83 3.2 MATERIALS AND METHODS ...... 85 3.2.1 Selection of Mother Plant ...... 85 3.2.2 Acemannan Isolation and Purification ...... 85 3.2.2.1 Plant collection for acemannan analysis ...... 85 3.2.2.2 Isolation and Purification ...... 86 3.2.3 Characterization and Qualitative Analysis ...... 86 3.2.3.1 Fourier Transform Infrared (FT‐IR) Spectroscopic Analysis ...... 86 3.2.3.1.1 Chemicals ...... 87 3.2.3.1.2 Instrumentation ...... 87 3.2.3.1.3 Sample Preparation ...... 87 3.2.4 Nuclear Magnetic Resonance Spectroscopy (NMR) ...... 87 3.2.5 Acemannan determination from Aloe vera calli under carbohydrate stress conditions. .... 87 3.2.6 TLC Analysis ...... 88 3.2.7 Recycling Preparative High Performance Liquid Chromatography ...... 88 3.2.7.1 Reagents and Chemicals ...... 88 3.2.7.2 Chromatographic Conditions ...... 89 3.2.8 Quantitative Analysis ...... 89 3.2.8.1 Instrumentation ...... 89 3.2.8.2 Sample Preparation ...... 90 3.2.8.3 Chromatographic Conditions ...... 90 3.2.9 Preparation of Standard Calibration Curve ...... 91
iv
3.3 RESULTS AND DISCUSSION ...... 92 3.3.1 Selection of Elite Mother Plant ...... 92 3.3.2 FT‐IR Spectrum Analysis ...... 93 3.3.3 Determination of Acemannan in conventionally propagated, in vitro regenerated Aloe vera plants and calli ...... 96 3.3.4 Identity assessment of acemannan by ¹H‐NMR ...... 98 3.3.5 Analysis of acemannan production and enhancement under different carbohydrate stress conditions ...... 101 3.3.6 Standard Calibration Plot ...... 106
CHAPTER 4 – BIOTRANSFORMATION
4.1 INTRODUCTION ...... 115 4.1.1 Biotransformation ...... 115 4.2 MATERIALS AND METHODS ...... 116 4.2.1 Establishment of Plant Cell Culture ...... 116 4.2.2 Growth Curve Analysis ...... 116 4.2.3 Biotransformation of Adrenosterone ...... 117 4.3 RESULTS AND DISCUSSION ...... 118 4.3.1 Suspension Cultures and Growth Curve Analysis ...... 118 4.3.2 Biotransformation of adrenosterone ...... 119
CHAPTER 5 – MOLECULAR ANALYSIS
5.1 INTRODUCTION ...... 122 5.1.1 Molecular Analysis of Aloe vera plants by Genetic/Molecular Markers ...... 122 5.1.2 Random Amplified Polymorphic DNA (RAPD) ...... 124 5.1.3 Inter Simple Sequence Repeats (ISSR) ...... 125 5.2 MATERIALS AND METHODS ...... 129 5.2.1 Evaluation of Genetic stability of Aloe vera plants by RAPD and ISSR Marker Assays ...... 129 5.2.2 Plant Material ...... 129 5.2.3 DNA extraction ...... 129 5.2.4 DNA quantification ...... 131
v
5.2.5 Primers ...... 132 5.2.6 Molecular Marker ...... 132 5.2.7 DNA Amplification by RAPD‐PCR ...... 132 5.2.8 Agarose Gel Electrophoresis ...... 133 5.2.8.1 Melting of the Agar ...... 134 5.2.8.2 Pouring the Gel ...... 134 5.2.8.3 Setting up the Gel Tank ...... 134 5.2.8.4 Running and Analyzing the Gel ...... 135 5.3 RESULTS AND DISCUSSION ...... 136 5.3.1 Evaluation of Genetic Stability of Aloe vera plants by RAPD Marker Assay ...... 136 5.3.2 Evaluation of Genetic Stability of Aloe vera plants by ISSR Marker Assay ...... 142 5.3.3 Combined Analysis of RAPD and ISSR Markers ...... 144
CHAPTER 6 – GENERAL DISCUSSION
6.1 GENERAL DISCUSSION ...... 147 6.1.1 Acemannan Enhancement ...... 147 6.1.2 Future Prospects ...... 150
REFERENCES / PUBLICATION / APPENDIX
REFERENCES ...... 152 PUBLICATION ...... 176 APPENDIX ...... 178 List of Tables ...... 179 List of Figures ...... 180 List of Graphs ...... 181
vi
CHAPTER 1
GENERAL INTRODUCTION
vii Chapter 1 General Introduction
1.1 GENERAL INTRODUCTION
1.1.1 Scope and Significance of Biotechnology
Biotechnology is a science of organized and measured application of biological agents for the purpose of valuable use. It is the unified utilization of microbiology, biochemistry, and molecular biology to acquire specialized and technological use of the competencies of biological agents. Hence, biotechnology has appeared as a branch of science with enormous prospects and potentialities for the human wellbeing with a wide scope of ranging from human health, food processing to environment protection. The following examples will highlight the importance of biotechnology in various streams:
1.1.1.1 Application of Biotechnology in Medicine
Biotechnology is used in producing the monoclonal antibody, RNA, DNA analyses to diagnose various diseases; the valued drugs such as interferon and insulin have been blended with bacteria for the treatment and cure of human diseases. Application of
DNA fingerprinting in identification and recognition of parents and criminals. The development of recombinant vaccines for the treatment of diseases such as human hepatitis B etc, through genetically engineered microbes are among the few in the long list of notable achievements.
1.1.1.2 Industrial Biotechnology
This is a field of biotechnology which was introduced on big scale for producing the alcohol & antibiotics through microorganisms. Still nowadays a range of chemicals and pharmaceutical drugs like glycerin and lactic acid are being prepared through
8 Chapter 1 General Introduction genetic engineering to have improved quality and volume. Biotechnology has delivered a quite effective, proficient and economical technology for producing large range of bio-chemicals like immobilized enzymes. The field of protein engineering is another significant section of biotechnology in which present enzymes and proteins are remodeled for the purpose of a particular function to improve the effectiveness and increasing the efficiency in their performance.
1.1.1.3 Biotechnology and Environment
Biotechnology has played a key role in the dealing with the environmental issues like depletion and exhaustion of natural resources for non-renewable energy, preservation of biodiversity, pollution control, etc. For example bacteria are being used to cleanse and detoxified industrial wastes, for the production of biogas and in battling against oil spills for treatment of sewage. Bio pesticides provide environmental friendly and safer substitute to chemical pesticides for controlling pests, insects and diseases.
1.1.1.4 Plant Biotechnology
In a situation where the growth of world population is outperforming the food supply, the plant biotechnology specially, is required to be applied immediately in all the spheres of life. The successes and achievements in plant biotechnology have already exceeded the entire previous anticipations and expectations and enhance the hopes of even more favorable and bright future. The revolution in the agricultural biotechnology rests on continuous, successful, innovative research and development activities, public acceptance and supportive regulatory climate.
The biotechnology in agriculture is fully unified and integrated with classical breeding and physiology which aims:
9 Chapter 1 General Introduction
For fast and economical clonal multiplication of forest trees and fruits as a
support to classical breeding.
For producing free of virus genetic stocks and planting material.
For unification of microorganisms into generation of engineered organisms and
agricultural production systems.
Safeguarding plants against the growing threats and dangers of biotic and
abiotic stress.
Development of innovative genetic variations through soma-clonal variation.
Expanding the prospects and horizons of biotechnology by producing specialty
foods, pharmaceuticals and biochemical.
The techniques of genetic engineering are used in producing transgenic plants
with necessary and desired genes such as herbicide resistance, disease
resistance, improved shelf life of fruits etc.
Molecular breeding has accelerated the process of upgrading and improvement
of crops for example, molecular markers such as RFLP, SSRs provides
powerful and effective tools for the indirect selection of both the qualitative &
quantitative aspects and traits and as well for studying the diversity of
genotypic.
1.1.2 Medicinal Plants
Medicinal plants are essential source of natural capital of a country and are considered to play a beneficial role in health care. They aid in providing raw materials for the production of traditional and contemporary medicines and also serve as important therapeutic agents. The system is in existence since ancient times and prospering today as the prime form of medicine. In 1999, Buckingham reported more than 200,000 species of higher plants in the world. World Health Organization (WHO)
10 Chapter 1 General Introduction presented a compiled report from literature of 90 countries, including the classical manuscript on Unani and Ayurvedic medicine which includes more than 20,000 species of medicinal plants. Norman Farnsworth, a pharmacognosist at the University of Illinois (USA), reported that there are 89 recommended plant-based drugs.
According to WHO, 80% of the world population in the developing countries rely on herbal therapies for their basic health necessities (Khan, 1985). Suitable medications are formulated by using either dried or a fresh part of plant (stems, leaves, roots, fruits, flowers or seeds). These preparations are compressed to make pills or tablets and also used to prepare various infusions (teas), tinctures, extracts etc., or used for cosmetic purpose in the manufacturing of creams, ointments and lotions. In last two decades there has been a tremendous escalation in research on medicinally important plants. A number of new medicines have been discovered with advancements in production technology to harvest pharmaceutically important metabolites. During this period there has also been an increase in research publication on medicinal plants.
Considerable amount of foreign exchange is being generated by the export of medicinal plants. Thus indigenous medicinal plants play a substantial role in the economy of a country.
11 Chapter 1 General Introduction
1.1.3 The Aloe vera Plant
1.1.3.1 Scientific Classification
Kingdom: Plantae-Plants
Sub kingdom: Tracheobionta-Vascular plants
Superdivision:Spermatophyta-Seeds
Division: Magnoliophyta-Flowering Figure 1: Aloe barbadensis plant
Class: Liliopsida
Sub class: Liliidae
Order: Asparagales
Family: Xanthorrhoeaceae
Genus: Aloe
Species: Aloe vera (L.) Burm.f. - Barbados aloe
1.1.3.2 Nomenclature
Barbados Aloe, Chinese Aloe, True Aloe, Indian Aloe, First Aid Plant, Wand of
Heaven, Burn Aloe and Miracle Plant are the widespread common names of this plant
(Jamir, 1999; Barcroft and Myskja, 2003; Liao, 2004; Ombrello, 2008).
According to most of the literature cited, the correct species name is Aloe barbadensis
Mill., and Aloe vera (L.) Burm. f. is regarded as a synonym. As per the International
Rules of Botanical Nomenclature, Aloe vera (L.) Burm. f. is the authentic name for this
12 Chapter 1 General Introduction species (Newton, L. E. 1979; Bruneton, 1995). According to the Angiosperm
Phylogeny group (APG) III system (2009), the genus has been assigned to family
Xanthorrhoeaceae. Previously it has also been placed in families Liliaceae and
Aloeceae.
1.1.3.3 Common or other Local, Multi‐lingual names
Barbados Aloe, Curacao Aloe, Indian Aloe, Jafarabad Aloe (English); Aloes, Aloes vulgaire (French); Echte Aloe (German); Ghrit Kumari, Kanya (Bengali & Sanskrit);
-Arabic); Kumarpathu, kunawar (Gujarati); Elva (Punjabi); Ghee) اﻷﻟﻮﻩ ﻧﺒﺎت , اﻷﻟﻮة kanwar, ghi-kuvar (Hindi); Kolasoar, Komarika, Kattavazha (Malayalam); Korphad
(Marathi); Bhottu-katrazhae, Chirukattalai, Kottaalai (Tamil); Kalabanda (Telagu).
1.1.3.4 Taxonomy and Etymology
The genus Aloe comprises of about four hundred species of succulent flowering plants. The genus is native to Africa and also commonly found in the Arabian
Peninsula. Some literature recognizes the white spotted form of Aloe vera as Aloe vera var. chinensis, (Gao, 1997; Wang et al., 2004) however, wide variation in species is present due to the leaf spots (Akinyele, 2007). Linnaeus in 1753 described the Aloe vera specie for the first time as Aloe perfoliata var. vera. In 1768 Nicolaas Laurens
Burman and Philip Miller again gave detailed description of this specie. On 6th April,
1768, Burman described the species as Aloe vera in Flora Indica, while the species was described by Miller, as Aloe barbadensis after ten days in the Gardener’s
Dictionary (Newton, 1979). Molecular fingerprinting techniques such as chloroplast
DNA sequencing and ISSR profiling, suggest a relatively close relation of Aloe vera to
Aloe forbesii, Aloe inermis, Aloe striata, Aloe perryi a species native to Yemen
(Darokar et al., 2003 and Treutlein et al., 2003). With the exception of South African
13 Chapter 1 General Introduction species, Aloe striata, these Aloe species are endemic to Yemen, Sudan and Sumalia
(Treutlein et al., 2003). Due to the insufficient evidence regarding natural population of the species, some authors propose that initially Aloe vera may be a hybrid (Jones,
2000).
1.1.3.5 Aloe vera Botanical Description
It is a succulent herb having a stout erect stem, which supports a compact rosette of rigid and fleshy leaves up to 50 cm long, having 8-10 cm wide base narrowing to a long slender pointed tip. Leaves have curved spines along their margins. Young leaves have white streaks and spots while mature leaves are green but white at the base where it clasps the stem. Flowers are borne in cylindrical terminal racemes on central flower stalks, 5-100 cm high. The yellow perianth is divided into six lobes, about 2.5 cm long, with scattered bracts. Each flower has six protruding stamens and three-celled ovary with long style. The capsules contain numerous angular seeds.
Forms of the species vary in sizes of leaves and colors of flowers.
1.1.3.6 Ecological Distribution
Aloe native to East and South Africa comprises of over 300 species of herbs, shrubs and trees, spread worldwide. The Aloe plant is grown in warm tropical areas and cannot survive cold climates. The natural range of Aloe vera is unclear though the species is found growing wild throughout North Africa in Algeria, Morocco and Tunisia, along with the Canary and Madeira Islands (Aloe vera, African flowering plants database, 2008). In 17th century the species was introduced to some parts of Asia including Pakistan, India, China and various parts of Southern Europe (Farooqi and
Sreeramo, 2001). The species is widely naturalized elsewhere, occurring in temperate and tropical regions of Australia, Nigeria, Paraguay, Portugal, Turkey and the USA. In
14 Chapter 1 General Introduction
Pakistan A. vera is often planted in gardens in Sindh, Punjab and Baluchistan. But conversely in Pakistan and India the plants have reddish flower color, which are different from the populations of A. vera growing in China, Turkey and Europe having yellow to pale yellow flowers. This matter needs further investigation (Rehman et al.,
2009).
1.1.4 Economic Importance
1.1.4.1 Medicinal Plants in World Market
Traditional medicinal practices are the major forms of healthcare in the world, particularly in the developing countries like Pakistan, India, Bangladesh and China.
The expanding populations and declining domestic supplies are necessitating an increased international trade in order to meet demand. In 1996, World Bank reported estimated figure of 5 trillion US dollars of trade in medicinal plants and their products by 2050 (FAO, 2007). China, Japan, France, Germany, Egypt, Italy, Spain, England and America are considered to be the largest global markets for Medicinal and
Aromatic plants (Source: United Nations Commodity Trade statistics Database 2007-
2008).
1.1.4.2 Current Status of Medicinal Plant Resources in Pakistan
Among developing countries of the world, Pakistan occupies an exceptional position, as it has tremendous potential regarding the heritage and diversity of medicinal plants due to variations in its geographic and climatic conditions. In Pakistan more than 6000 flowering plants and a vast number of drug plants have been reported to occur in the northern and northwestern parts of the country (Ali and Qaiser, 1986). Medicinal plants commercially exploited in large quantities mainly occur in four ecological regions of Pakistan i.e. Alpine and high altitude regions, temperate and sub-tropical
15 Chapter 1 General Introduction forests, arid and semi-arid scrubs (Rafiq, 1998). Hussain, (2004) reported that in
Pakistan about 700 plant species are being used as medicinal and aromatic plants.
He further estimated that there are 25,000 plant species in the Himalayas, Hindu Kush and Karakorum regions, of which nearly 10,000 plants are beneficial. About 60 percent of the population in Pakistan relies on herbal remedies. More than 300 medicinal plants are found growing in the wild. Around 100 of these are collected and supplied by hakims and plant dealers and twenty species are traded nationally in the markets of Karachi and Lahore. According to the National Institute of Health (N.I.H) over 500 plant species are employed broadly in traditional medicines. Tibbi
Pharmacopoeia of Pakistan (Pharmacopoeia of Traditional Drugs compiled by the
Tibbi Board) has listed around 900 single drugs and about 500 compounds preparations made out of medicinal plants. In Pakistan there are about 30 big herbal manufacturing companies. The number of herbal medicine producers in private sector run into hundreds. The annual turnover of some large herbal companies can be compared to international manufacturers in Pakistan. In the global market Pakistan ranks among the top ten exporting countries of medicinal and aromatic plants in the world, exporting plants worth US$ 5.45 million per year. Over 60% of the total export originates from the Hindukush-Himalayas regions of the country (Champion et al.,
1965; Sher et al., 2005; Sher & Hussain, 2009). The destinations of such exports include Germany, USA, Middle East, India, Iran, etc. However, Pakistan also imports some of the medicinal and aromatic plants worth US$ 130 million from the above countries (Sher & Hussain, 2009). Such imports have increased over the last ten years.
16 Chapter 1 General Introduction
1.1.4.3 Aloe Trade
1.1.4.3.1 Current Market Scenario
Aloe vera is among the most vital ingredients in the medicinal and cosmetics industry at present. The Aloe extracts are applied in moisturizing preparations (Barcroft, 2003).
A. vera is among the highly commercial Aloe species and treatment of leaf pulp has turned into huge industry globally. In food and beverage industry, it is applied as the basic functional foods as well as a separate ingredient in other food items, for producing beverages and health drinks containing gel. In the toiletry & cosmetic industry, it has been applied as basic material for producing lotions, creams, facial cleansers, shampoos, soaps and other products. In pharmaceutical and medicine industry, it has been utilized for making the topical products like gel and ointment preparations, and also in the manufacturing of capsules and tablets (Eshun et al.,
2004). Aloe vera plant has also gained the “Award of Garden Merit” of Royal
Horticultural Society (RHS Plant Selector Aloe vera AGM / RHS Gardening, 2012).
1.1.4.3.2 Raw Material
The worldwide gross revenue of raw Aloe leaves add up to US$ 60-90 million dollars, which is projected to rise at a rate of 40% in the next five years (Grindlay and
Reynolds, 1986).
1.1.4.3.3 Present Global Players
Global market for Aloe based value added, final products is around US$ 110 billion,
(IASC, 2004). USA is the major supplier of Aloe, having more than 50% share in the global market. Whereas South America and Mexico contribute to 20-30% of herbal supplies and Australia, China, India and Pakistan collectively have a market share of only 10%. Keeping in view the increasing worldwide demand, cultivation of this herb
17 Chapter 1 General Introduction must be encouraged. This will also help Pakistan in earning substantial foreign exchange and ensures financial stability (Source: IASC market report).
1.1.5 Medicinal Importance
1.1.5.1 Use of Aloe in Traditional Therapy
Aloe has been used since prehistoric period for the cure of various ailments. The word
“Aloe” is derivative of the Arabic word “Alloeh” meaning shiny and bitter substance
(Tyler et al., 1976). For centuries products of Aloe vera have been used in the manufacturing of medicines and cosmetics (Eshun, 2004; Marwat, 2011). The genus
Aloe contains more than 300 different species with Aloe aborescens Aloe barbadensis
Miller (Aloe vera) and Aloe chinensis being the most popular. Aloe barbadensis Miller is recognized as most active biologically (Joshi, 1997; Yagi et al., 1998; WHO, 1999).
Species of Aloe vera used as folk medicine include Cape Aloe (Aloe ferox), Curacao
Aloe (Aloe barbadensis or Aloe vera) and Scocotra Aloe (Aloe perryi). For thousands of years several ancient civilizations like Egypt, Greek, Africa and India have documented the virtues of Aloe vera (Rolf and Zimmerli, 2000). The earlier descriptions of the use of Aloe vera as traditional remedy goes back to 1500 B.C. being described in the Papyrus Ebers and Sumerian Clay Tablets, stored in Leipzig
University (Barcroft and Myskja, 2003). In early records of 1750 B.C. findings of Aloe vera carvings in cave walls reveal it to be used by ancient Egyptians in preservatives
(Shelton, 1991; Longe, 2005).
The first detailed record of the pharmacological effects of Aloe is noted in “The Geek
Herbal” by Dioscorides in the 1st century A.D. (Barcroft and Myskja, 2003). This document gives twelve formulations for mixing Aloe with other reagents for the
18 Chapter 1 General Introduction treatment of both internal and external human disorders. It is also mentioned that
Cleopatra used Aloe gel to keep her grandeur (Longe, 2005). According to the ancient records, Socotra Island was conquered by King Alexander to obtain Aloe plants for the treatment of wounded soldiers. This demonstrates that the medicinal and cosmetic value of the plant has been recognized and availed since centuries.
1.1.5.2 Use in Modern Medicine
The healing miracles of Aloe vera has been well-known all over in the history of the world for thousands of years, but only in the last few decades recent advancement in medicine and laboratory investigations have disclosed the amazing potential of this miraculous plant. Prof. Rowe (1941), was the pioneer in investigating the A. vera plant and his efforts lead to its first detailed chemical evaluation. Dureja in 2005 and Park in
2006, reported Aloe vera to contain 200 active compounds and more than 70 nutrients including saponins, sugar, anthraquinones, enzymes, vitamins, amino acids, minerals and salicylic. The Aloe vera plant has been subjected to countless studies due to its unique healing abilities and multiple pharmacological effects. There have been many studies on Aloe vera. Researchers have isolated and characterized numerous biologically active compounds from all parts of this plant. Saccu and Procida in 2001 found that Aloe leaf can be divided into two main parts known as Aloe gel and Aloe latex. Aloe latex also known as Aloe juice is yellow color bitter substance obtained from the pericyclic tubules in the exterior skin of the leaves. The main active constituents of Aloe latex are hydroxyanthracene derivatives (15–40%) like glycosides, anthraquinone, aloin A and B (Saccu, Bogoni & Procida, 2001). Aloe latex is famous for cathartic properties. Aloe vera has also played a significant role in guarding from oxidative stress (Barcroft 1999; Foster, 1999; Joseph and Justin, 2010;
El-Shemy, 2010). Scientists have discovered that Aloe vera can also prevent the risk
19 Chapter 1 General Introduction of heart diseases. Patients given Aloe vera showed a significant improvement in their lipid profile, reducing the levels of total cholesterol, phospholipids, triglycerides, which if elevated increase the risk of cardiovascular disorders (Balch and James, 2000;
Joseph and Justin, 2010).
In the inner part of the fresh leaves, colorless gel is present called Aloe gel (Reynolds
& Dweck, 1999). The gel contains mainly water (>98%) and polysaccharides
(hemicellulose, pectins, cellulose, glucomannan, acemannan and mannose derivatives). Acemannan is recognized as key functional component in Aloe vera and is comprised of a lengthy chain of acetylated mannose (Femenia et al., 1999; Djeraba
& Quere, 2000; Lee et al., 2001). Aloe gel is commonly commercialized as powdered concentrate. Chois and coworkers in 2001 reported that Aloe vera gel holds a tremendous potential to heal wounds. It has also exhibited anti-bacterial, anti-fungal activities (Reynolds, 1999; Fatima habeb et al., 2007; Muhammad et al., 2011). Davis and others in 1994 demonstrated its anti-inflammatory action and it is also possesses immune stimulating activities (Womble et al., 1992; Stuart et al., 1997). It was found to stimulate fibroblasts, the skin cells recognized for healing wounds and manufacturing of collagen protein which controls the processes of wrinkling and aging of skin
(Joseph and Justin, 2010). It is used in the therapy of gastrointestinal ulcers (Blitz et al., 1963), diabetes mellitus (Hidahiko Beppu et al., 2006), and also found to be active against cancer (Langmead et al., 2004; Akevn, 2007). Researchers found that Aloe vera can inhibit HIV-1 in the AIDS patients it has even secured and protected their immune system from harmful and toxic side effects of Azidothymidine, the first approved treatment of HIV (Urch and David, 1999). Research on the medicinal properties of Aloe has expanded in scope during the last three decades and the
20 Chapter 1 General Introduction production or transformation of value added compounds, which have pharmaceutical significance, is still needed to be explored.
1.1.6 Components of Aloe vera and their biological effects
The Aloe vera plant contains over 200 bioactive compounds and 70 nutrients, including 20 minerals, 12 vitamins and 18 amino acids.
21 Chapter 1 General Introduction
1.1.6.1 Pharmaceutical Activities of Aloe vera Components
Table 1: Showing the pharmaceutical activities of Aloe vera components
Components Pharmaceutical Activities
Glycoprotein Cell proliferation activity, wound healing effect, anti-allergy. Barbaloin Purgative.
Aloe-emodin, emodin Purgative, cell proliferation, anti- cancer, anti-protozoa, anti-bacteria, anti-oxidant, genotoxicity, mutagenicity. Mannose-6-phosphate Wound healing, anti-inflammation.
Polysaccharide Anti-cancer, immunomodulation.
Acemannan Immunomodulation,anti-microbial effect, anti-tumor.
Aloesin Cell proliferating activity, melanin synthesis inhibition.
Β-sitosterol Anti-inflammation activity, angiogenesis.
Diethylhexylphthalate Anti-cancer.
Low molecular weight compounds of Immunomodulation. 0.5_1 kDa
(Seminars in Integrative Medicine, Vol. 1 (March), 2003, pp. 57)
22 Chapter 1 General Introduction
1.1.7 Propagation Techniques
1.1.7.1 Cultivation Practices
Aloe vera is a succulent, perennial plant having short stem (30-100cm) and shallow roots. Aloe does not have a true stem but produces bloom stalks. The plant generally grows close to the ground in a typical rosette shape. The fleshy leaves about 50 cm long and 10 cm wide having thickness of 1.0-2 cm. Leaves form a densely crowded rosette having spiny margins. The plant is in leaf all year round.
Soil: It is a strong and hardy plant and prefers growing in sandy and loamy soils, but can also tolerate salinity and nutrition deficient soils. Sandy coastal to loamy soils of the plains with a pH up to 8.5 is most suitable for their growth.
Climate: The plants are cultivated between March and June. It is widely adapted to humid and warm climatic conditions, with even 150-200 cm to about 35-40 cm of yearly rainfall during growing period. However, in dry regions, the crop should be provided with protective irrigation. The species is hardy in zones 8–11, although it is intolerant of very heavy frost or snow (British Broadcasting Corporation, 2008).
Land Preparation: The land should be ploughed twice before cultivating plants and the plantation area should be clear of weeds. Minor canal system can also be constructed for irrigation purpose. About 30 t/ha of farm-yard-manure is also added at the time of land preparation.
Propagation: The plants are generally propagated by root-suckers, rhizome-cuttings or seeds.
Seed: Seeds are sown in spring season and kept under greenhouse conditions at
16ºC. Seed germination usually takes 1-6 months. Then the seedlings are carefully
23 Chapter 1 General Introduction planted in individual pots having well drained soil and gown under the sunlit area of the green house for their first 2 winters. If growing the seeds outside, then they are planted in early summer season to become well established till the arrival of winter. In winter suitable shelter is provided due to the harsh cold weather. By spring season divisions of offshoots are available. The plants frequently produce offshoots which can be separated at any time of the year. Warm conditions are suitable for root production and plant re-habituation.
Cuttings: When there is a need to produce buds in large quantities, the adult plants are decapitated at about 10 cm above the ground level and all the leaves and buds are removed. After two months numerous buds about 5 cm long will arise. When these buds grow up to 10 cm height, they are cut off and sown to produce plantlets.
After 3 months about 20 cm tall plantlet will be ready for planting in the field at a spacing of 80 cm x 60 cm or at the rate of over 2,000 plantlets per ha. Plantation may be done the year round. These plantlets are drought resistant and can grow in any type of soil excluding swamps. They prefer poor, dry, sandy and well-drained soil
(Fuentes et al., 2000). Soil preparation does not require any special modifications and any growth system, conserving the soil structure and ensuring appropriate rooting, shooting and plant development can be selected.
Flowering: Flowering starts by the end of December to early January and into March, but there is rare formation of fruits. Three months later dehiscence of fruits is observed in the mature dry capsules. The flowers are androgynous i.e having both male and female organs. The color of the flowers varies from species to species which may be red, orange, yellow etc.
24 Chapter 1 General Introduction
Manuring: Fertilization is done with good supplies of a mixture of 150 kg/ha of NPK
(Nitrogen, Potassium and Phosphorous), or alternatively with some 30 to 50 tons of organic matter per hector is advised. Then after establishment of plants, fertilizers are applied near the roots of the plant in the soil.
Irrigation: Irrigation of the land is completed immediately after plantation, according to the moisture content of the soil. However water should not be allowed to stagnate near the plant.
Weeding: Land must be kept weed free and weeding should be practiced twice a year.
Harvesting: Harvesting is usually done after two years of plantation and then after every six months. Harvesters are recommended to wear shirts with long sleeves and gloves for protection. Harvesting is done manually and knives are used to make incisions at the extreme base of the oldest leaves. Then 6-8 leaves per plant are cut and separated (Martinez et al., 2000). Then new sprouts arise in the spring season from the rhizome portion left in the soil. These can be utilized as planting material for the successive crop. Commercial yield of the crop can be obtained from 2- 5 years, after which replantation is done. Reported average yield is 40t/ha leaves per harvest
(Martinez et al., 2000).
Pests: The species is resilient to most of the insect and pests. However some aphid species and bugs may cause damage to plant growth and development (Florida
Department of Agriculture & Consumer Services, 2008).
25 Chapter 1 General Introduction
1.2 Plant Tissue Culture Technology
1.2.1 Brief History
The science of growing plant tissues, cells or organs separately from mother plant on artificial nutrient media, in a controlled environment is called plant tissue culture
(George, 1984). Although strictly it refers to the culture of an undifferentiated mass of plant cells only (Duncan and Widholm, 1986; Street, 1977; George and Sherington,
1984). Plant tissue culture technology has become a powerful tool in providing solutions to many fundamental and applied problems in plant biotechnology. Living organisms vary greatly in their complexity, life-cycles, and mode of propagation and reproduction. Regardless of these differences, such organisms possess or have possessed during their long evolutionary history, the common feature of being represented at some stage by a single cell. All complexities and events occurring during their life-cycle must unfold from this single unit. The cell must therefore contain all the information necessary for the organism to grow and reproduce in its environment and thus can be called totipotent.
Two German biologists, M.J. Schleiden and T. Schwann, in 1838 put forward the first theoretical attempt to explain the complexity of multicellular organisms (Thomas and
Davey, 1975). Although they realized that complex interactions must occur between different cells, tissues and organs, they suggested that each cell is an independent unit capable of forming a new organism. They clearly implied that differentiated cells of a multicellular organism still retain the information which was present in the first single cell, the fertilized egg.
26 Chapter 1 General Introduction
According to Thomas and Davey (1975), Haberlandt was the first to realize the importance of totipotency of plant cells in 19th century and enunciated ways in which this property, if it existed could be exploited. As Haberlandt mentions, "That he is not aware of any systematic and organized efforts were made to culture from higher plants, separate vegetative cells into basic nutrient solutions. Although the outcome of these culture experiments provide some exciting and fascinating insights to the potentialities and properties of a cell as a fundamental unit. Further, it would present knowledge regarding the corresponding and inter-relationships impacts which exposes the cells within multicellular organisms. He finally concluded as "Without allowing me to ask more questions, I understand, that I am not presenting too bold claims if I show to the prospect that, in this way, one could effectively develop synthetic embryos through vegetative cells".
In experimental plant tissue culture the first major steps were made in 1922 by W.J.
Robbins in America and by W. Kotte, a student of Haberlandt (Thomas and Davey,
1975). They used root meristems obtained from grass seeding. When the excised root tips were cultured in liquid nutrient media containing inorganic salts and glucose, vigorous rooting was observed. Unfortunately, the growth of these roots rapidly declined in culture, and virtually ceased, even if meristems were excised and transferred to fresh medium. The cultural conditions clearly did not permit continuous growth. However, these studies laid the foundation of plant tissue culture as it exists today.
The first successful cultures were those of P.R. White, who, in 1934, isolated and grew excised root tips of tomato plant for prolonged periods, in liquid nutrient medium composed of sucrose, inorganic salts and yeast extract. Since then many researchers
27 Chapter 1 General Introduction have tested and improved the environmental and nutritional necessities of plant cells in culture, through the work of Murashige and Skoog (1962), which laid the foundation of most tissue culture media used today.
In 1974, Murashige exhibited that physical and nutritional parameters can affect the plant regeneration process and in 1948 Skoog and his team investigated about the cytokinins and the hormonal control of shoots and root regeneration through tobacco callus has set-up the basis of manipulating organ initiation and given the principle on which all micropropagation is based. A significant development was made in 1960 in the culture of single cell, when Bergmann reported that it was possible to get cell suspension with a large proportion of single cells using a simple filtration method to eliminate the larger cell aggregates. The single cells can be induced to grow by mixing with agar and cultured in petri dishes. Later, it was observed that the calli can be induced to produce embryoides obtained through fully differentiated leaf cells. The capability to culture single cells offers opportunities to isolate normal and genetically modified single cells.
Plant tissue culture is a vital element and pillar of plant biotechnology. The principle involved in plant tissue and cell culture define the competency to develop, regenerate and propagate plants from a single cell, tissue and organ under semi controlled, manipulative and sterile environmental conditions in a chemical nutrient medium of known composition, (Murashige & Skoog). The technique of growing plant cells separately from an intact plant is crucial in many areas of plant sciences.
Tissue culture is based on the principle of totipotency, which means that every living cell has the genetic potential to reproduce the entire organism. The term micro- propagation is specifically used in terms of the tissue culture techniques applied "to
28 Chapter 1 General Introduction the propagation of plants beginning with the isolation of very small plant part (explant) from an intact plant, grown aseptically in a container with rigidly controlled nutrition and environment, in which it can express its intrinsic or induced potential”, (Dixon,
1987; Smith and Drew, 1990). In practice, many propagators use the term micropropagation and tissue culture interchangeably to mean any plant propagation procedure utilizing aseptic culture. A synonymous term is in vitro culture. In a plant tissue culture laboratory the expenditure of much of the equipment is targeted at careful control of all the components (eg. media components, light, atmosphere and temperature regimes) pertaining to the physical and physiological environment of the system.
1.2.2 Explant Source
Plant tissue culture begins with the excision of a small piece of plant followed by sterilizing and placing it into aseptic culture. This small plant tissue known as
“explant” is an initiation point for all tissue cultures. It can be started from any part of plant i.e stem, root, leaf, nodal buds; however the success of any one of these varies among the species depending on the type, position and age of explants (Gamborge et al., 1976). Generally juvenile tissues give best response. Because all plant cells do not possess the same ability to express totipotency (Staba et al., 1980).
1.2.3 Nutrient Media
Other than the source of explants, optimizing the cultures physical environment and chemical composition of the culture medium preferable to different plant species, is nearly the main task for the establishment of a successful plant cell and tissue culture technique. These factors have a major effect on the regeneration ability, propagation rate and nurturing of new plants into the culture system.
29 Chapter 1 General Introduction
The selection of the tissue culture medium mainly rest on the types of species required to be cultured. For example certain species are delicate and sensitive to high salts or have varied PGRs requirements. Certain tissues are better responsive to solid medium whereas others adopt a liquid medium. Hence the growth and development of culture medium designs is the outcome of methodical trials and testing keeping in vice particular needs and requirements of a specific culture system. Initially among the originally formulated plant tissue culture media for root culture is the White’s medium.
For the regeneration of plant from tissues and callus, Murashige and Skoog (MS) medium is the most appropriate and generally used medium. Because of the content of potassium and nitrogen salts, this medium is a high salt medium. Because of lesser quantity of nitrate particularly ammonium salts than MS medium, B5 medium works good for protoplast culture. The salt concentration in Nitsch’s medium prepared for another culture is intermediate to MS and White.
Successful regeneration is ensured by providing the tissue material with vital growth elements in the form of macronutrients, micronutrients, vitamins, amino acids or nitrogen supplements, growth regulators and addition of carbon in the organic form as sugar. The International Association for Plant Physiology defines the elements required in concentrations greater than 0.5 mM/L as macronutrients and those required less than 0.5 mM/L as micronutrients (De fossard R., 1976). However, for achieving maximum growth rate the optimal concentration of each nutrient varies among plant species.
1.2.4 Plant nutrients and their role in plant tissue culture
In case of intact plants they can generate their own food however in the in vitro cultured plant parts or cells needs a range of nutrients and appropriate physical
30 Chapter 1 General Introduction conditions for their development and growth. The design and composition of plant tissue culture medium rests on the nature and type of plant tissues or cells that are used for culture. There is no single medium which can be used for all types of organs and plants, therefore the formation and composition of the culture medium for each plant material has to be worked out. In the plant development and survival, there are sixteen chemical elements which are considered to be vital. Deprivation of even one of these essential elements cause physiological disorders or even cell death (Koshiba et al., 2009). These chemical elements are classified into two categories: non-mineral elements and mineral elements.
1.2.4.1 Non‐mineral Elements
The non-mineral nutrients are carbon, oxygen (O) and hydrogen (H). These nutrients are present in water and air. Plants use energy from the sun to convert carbon dioxide and water into sugars and starches in a process called photosynthesis, which are utilized as plant food.
1.2.4.2 Mineral Elements or In‐organic Supplements
The 13 other essential nutrients required by plant are termed as mineral elements which are separated into two categories: macronutrients and micronutrients.
1.2.4.2.1 Macronutrients
The mineral nutrients needed in a concentration greater than 0.5 mmoles/L are referred to as macronutrients. These constitute of six main elements: phosphorous, nitrogen, potassium, magnesium, calcium, and sulphur present as salts in different media. Macronutrients are further separated into two subgroups: primary and secondary nutrients.
31 Chapter 1 General Introduction
Primary nutrients are nitrogen (N), phosphorous (P) and potassium (K). These major nutrients are utilized by plants in larger volumes, so they are often provided in the form of fertilizers in soil. Culture media should contain at least 25 mmoles/L of nitrate and potassium. In 1982, Hyndman and his team demonstrated that decrease in the concentration of ammonium nitrate and sodium nitrate was the decisive factor in improving the rooting percentage in micro shoots of rose plants.
Secondary nutrients are calcium (Ca), magnesium (Mg) and sulphur (S). They are usually required in scarce amount and not supplemented as fertilizers. The optimum concentrations of Mg, S and Ca range from 1-3 mM if other requirements for cell growth are provided (Torres, 1989). The salts normally provided in tissue culture also provide sodium and calcium.
1.2.4.2.2 Micronutrients
The inorganic elements which are essential for plant growth but required in small quantity, less than 50 mmoles/L constitute the micronutrients. They include iron, manganese, boron, zinc, copper and molybdenum. Of these elements, iron is the most critical and its chelating forms are used in the preparation of tissue culture media. Usually iron chelated to EDTA is used, which should be used in low concentrations, as it is the cause of genetic aberrations in plants. Murashige in 1979 reported that relatively low concentrations of salts in the medium are known to enhance rooting of micro shoots.
32 Chapter 1 General Introduction
1.2.4.2.3 Organic Supplements
1.2.4.2.3.1 Vitamins
Vitamins are synthesized endogenously by plants and used as catalysts in various metabolic processes. However, in plant cell cultures vitamins are not synthesized in the optimal concentration to support plant growth. Thus an exogenous supply of vitamins is provided through nutrient medium to achieve optimal plant development.
The most frequently used vitamins are Thiamine (Vitamin B1), Nicotinic acid (Niacin or
Vitamin B3), Pyridoxine (Vitamin B6), Pantothenic acid (Vitamin B5) and myoinositol.
Thiamine imposes significant application in tissue culture media as it functions as a coenzyme in the organic acid cycle of respiration (Krebs cycle or citric acid cycle), thus necessarily required by all cells for growth (Ohira et al., 1976). Niacine is an active component of coenzymes involved in light energy reactions, while pyridoxine also serve as coenzyme in various metabolic pathways and stimulate growth.
Vitamins in combination with other media components also effect callus production, somatic embryogenesis, rooting and embryo development (Abrahamian et al., 2011).
The other types of vitamins like ascorbic acid, biotin, folic acid, tocopherol (vitamin E), pantothenic acid, riboflavin, p-amino-benzoic acid are used in certain cell culture media but they are not the limiting factors of growth.
1.2.4.2.3.2 Amino Acids
The necessary amino acids required for optimum growth are normally made by most of the plants, but the inclusion or addition of some amino acids or amino acid mixtures is specially vital for building cultures of protoplasts and cells. Nitrogen is provided by amino acids as the source as to plant cells which is conveniently assimilated by cells and tissues much quickly than the sources of inorganic nitrogen. The mixtures of
33 Chapter 1 General Introduction amino acids like L-glutamine, casein hydrolysate, adenine and L-asparagine are regularly applied as the sources of organic nitrogen in culture media. Casein hydrolysate is normally applied at concentrations in between 0.25-1 g.l-1. The amino acids utilized for boosting and enhancement of the growth of cell in culture media comprised of; glutamine up to 8 mM, glycine at 2 mg/L, L-arginine, asparagine at
100mg/L, and L-tyrosine at 100mg/L and cysteine at 10 mg/L (Torres et al., 1989).
1.2.4.2.3.3 Carbon and energy sources
Sugar is a very important energy source and a vital component of the nutrient media.
Since most in vitro plant cultures are unable to photosynthesize effectively, leading to inadequate development of cells and tissues, limited gaseous exchange and lack of chlorophyll in tissue culture vessels. Hence they lack the auxotrophic ability and require external carbon as an energy source. The most preferred carbon or energy source used in tissue culture media is sucrose at a concentration of 20-60 g/L. When the nutrient media is autoclaved, sucrose is hydrolyzed to glucose and fructose which are utilized for growth. However, fructose if autoclaved has toxic effects.
In addition to sucrose in plant cell culture media, there are additional carbohydrates which are used as well. Among these comprise galactose, lactose, starch and maltose but they were described to be less efficient and effective as compare to either glucose or sucrose. Likewise glucose was more useful and effective as compare to fructose keeping in mind that glucose is used initially by the cells followed by fructose. It was repeatedly determined that autoclaved sucrose was superior for development than filter sterilized sucrose. Sucrose was noted to act as morphogenetic trigger in the construction and creation of auxiliary buds and branching of adventitious roots
(Vinterhalter et al., 1997). In the root induction the sucrose concentration plays a vital role and acts as a booster to osmotic potential. Commonly sucrose (3%) is used as a
34 Chapter 1 General Introduction carbohydrate source. In general Murashige and Skoog (1962) also mentioned that the use of 3% sucrose is better than 2% or 4% for the purpose of tissue culture. On a high osmolarity medium Lakes and Zimmerman (1990) reported the largest rooting percentage in apple.
Myo-inositol is added to activate the growth of cells of maximum species of plants
(Vasil et al., 1998). Although it is carbohydrate instead of vitamin, in small quantities myo-inositol is considered to act in cell division due to its split into pectin and ascorbic acid and integration into phosphatidyl-inositol and phosphoinositides.
1.2.4.2.4 Natural substances and extracts
Certain media were complemented with extracts or natural substances like hydrolysates, protein, coconut milk, yeast extract, malt extract, ground banana, orange juice and tomato juice to assess their outcome on the increase in growth.
These composite and complicated organic supplements had advantageous effects on in vitro plants growth. Therefore, a large variety of organic extracts are normally added now to culture media for the purpose of enhancement of various plant cells and tissues (Molnar et al., 2011).
1.2.4.2.5 Activated charcoal
The activated charcoal is often included to culture media which may have either a deleterious or beneficial effect. Differentiations and growth were activated in orchids
(Wang et al., 1976), carrots and onions (Fridborg, 1975), tobacco (Anagnostakis,
1974). An inhibition of cell growth on the other side was noticed on inclusion of activated charcoal to soybean culture medium (Fridborg et al., 1978). The reasoning of the action regarding activated charcoal was on the basis of adsorption of inhibitory
35 Chapter 1 General Introduction compounds from the medium, from the culture medium or darkening of the medium adsorption of growth regulators (Pan and Staden, 1998). In the medium the existence of 1% activated charcoal was determined to essentially upsurge hydrolysis of sucrose during autoclaving which result acidification of culture medium (Druart and Wulf,
1993).
1.2.4.2.6 Anti‐browning compounds
Numerous plants are rich in polyphenolic compounds. Subsequent to tissue damage at the time of dissection, these compounds will be oxidized through polyphenol oxidases and the tissue will turn into brown or black. The oxidation products are known to prevent enzyme activity, kill explants, and darken the tissues and culture media, a process which severely affects the establishment of explants. Activated charcoal at concentrations of 0.2 to 3.0% (w/v) is applied where phenol-like compounds are a trouble for in vitro growth of cultures. It can adsorb toxic brown/black pigments and also stabilize pH. Besides activated charcoal, polyvinylpyrolidone (250-
1000 mg/L), citric acid and ascorbic acid (100 mg/L each), thiourea or L-cysteine is also applied to avoid oxidation of phenols.
Some of the procedures used by various workers to combat this problem of browning are: (i) Adding antioxidants to culture medium, ascorbic acid, citric acid, polyvinylpyrolidone (PVP), dithiothreitol, bovine serum albumin, etc. (ii) Pre-soaking explants in antioxidant before inoculating into the culture medium; (iii) incubating the early stage of primary cultures in decreased light or darkness because it is known that phenolic oxidation products are formed under illumination; and (iv) Frequently transferring explants into fresh medium whenever browning of the medium is observed (Druart and Wulf, 1993).
36 Chapter 1 General Introduction
1.2.4.2.7 Plant Growth Regulators
Plant hormones are regulators formed by plants which in low concentration regulate physiological plant processes and move within plant from a site of production to site of action. The phytohormones have five clases: gibberellins, auxins, abscisic acid, cytokinins, and ethylene. The two main categories of growth regulators of special importance in plant tissue culture are auxins and cytokinins and while others are of minor importance, viz., gibberellins, abscisic acid, ethylene, etc. Some of the naturally- occurring growth regulators are indoleacetic acid (IAA), an auxin and zeatin and isopentenyl adenine (2 iP) as cytokinins, while others are synthetic growth regulators.
1.2.4.2.7.1 Auxins
The plant hormone auxin was discovered 70 years ago, although the concept of plant hormones and speculation over their roles in plant development were conceived much earlier (Reviewed by Abel and Theologis, 2010). Auxin was discovered following experiments on the reaction of coleoptile curvature in Gramineae. It owes its name to its effect on the elongation of cells (auxesis). Auxins have an indole nucleus with the basic formula C10H9O2N. A common feature of auxins is their property to induce cell division and cell elongation. The stimulation of division of cells of cambial origin resulted in initial successes with in vitro cultures. This effect leads to a number of cells, which further result in the formation of callus. Auxin has a clear rhizogenic action, i.e. induction of adventitious roots. It often inhibits adventitious and auxillary shoot formation. At low auxin concentration, adventitious root development dominates, while at high auxin concentration, the occurrence of root formation fails and callus formation appears. All the plants synthesize auxin that is modulated according to the stage of development. Auxin is present in sufficient concentration in the growing shoot tips or flowering tips of plants to ensure cell multiplication and elongation. Auxin
37 Chapter 1 General Introduction circulates from the top towards the base of the organs with a polarity strongly marked in young organs. The compounds most frequently used and highly effective are: naphthalene acetic acid (NAA), indole acetic acid (IAA), indole butyric acid (IBA), 2,4- dichlorophenoxy acetic acid (2,4-D). Other auxins in use are 2,4,5-trichlorophenoxy acetic acid (2,4,5-T), p-chlorophenoxy aceticacid (pCPA) and pichoram (4-amino-
3,5,6-trichloropicolinic acid).
1.2.4.2.7.2 Cytokinins
Cytokinins are a group of basically varied N6-substituted purine derivatives which have capability of inducing plant cell division. In 1950s the initial focus on a synthetic compound called kinetin, derived from autoclaved salmon sperm DNA has led to the discovery of cytokinins by Folke Skoog and colleagues (Miller et al., 1955). Later on
Miller, Skoog, and others noted that cytokinins are naturally occurring plant hormones that are vital for standard plant growth and development. In a series of classic experiments, Skoog and Miller in 1957 displayed that the ratio of cytokinin to auxin in nutrient media strongly affects the morphogenesis of roots and shoots in plant tissue culture. Several plant development procedures have been established to be affected by cytokinin, comprising chloroplast development, mobilization of nutrients, prevention of leaf senescence, root and shoot branching and cell expansion (Reviewed by Mok,
1994).
Cytokinins normally applied in culture media comprise 2iP (6- dimethylaminopurine),
Zeatin (6-4-hydroxy-3-methyl-trans-2-butenylaminopurine), BAP, kinetin (N-2- furanylmethyl-1H-purine-6-amine) and TDZ (thiazuron-N-phenyl-N-1,2,3 thiadiazol-
5ylurea). Cytokinins demonstrated to stimulate the cell division in culture media.
Retard root formation and induce shoot formation and axillary shoot proliferation. 2iP and Zeatin are naturally occurring cytokinins and zeatin is more effective. Zeatin, the
38 Chapter 1 General Introduction first endogenous cytokinin, was identified in 1963 in immature embryos of maize, while 2 iP was discovered a little later from plants attacked by the bacterium
Cornyebacterium fascines. Stock solutions of IAA and kinetic are stored in amber bottles or bottles covered with a black paper and kept in the dark since they are unstable in light.
In culture media the cytokinins are comparatively steady compounds with capacity of storage desiccated at -20ºC. It is frequently noted that cytokinins are difficult to dissolve and normally the inclusion of some drops of 1N HCl or 1N NaOH helps in their dissolution. Without causing injury and damaging the plant tissue, cytokinins can be dissolved in little quantity of dimethylsulfoxide (DMSO) (Schmitz, et al., 1972).
Being sterilizing agent DMSO have an extra advantage and can be directly included to the sterile culture medium having DMSO stock solutions.
1.2.4.2.7.3 Gibberellins
Gibberellins consist of over twenty compounds, of which GA3 is the most regularly used gibberellin. These compounds boost the growth and progress of callus (Vasil and Thorpe, 1998) and aid the elongation of dwarf plantlets (Torres, 1989).
1.2.4.2.7.4 Abscisic acid
To the plant tissue culture media, other growth regulators are often included for example, abscisic acid. Abscisic acid is a compound which is generally supplemented to activate the callus growth, according to the type of species. It boosts and improves shoot multiplying & restricts the development of embryo at later stages (Anagnostakis,
1974). Although the growth regulators are very costly medium ingredients, they do not
39 Chapter 1 General Introduction affect he overall cost of the medium grossly as their requirement in very small concentrations (Prakash et al., 2002).
1.2.4.2.7.5 Ethylene
Ethylene was identified long time ago as the only gaseous hydrocarbon hormone, but its functions as a growth regulator were not evident. The ethylene plant hormone is used in various physiological processes, involving plant development, growth and senescence. Ethylene also extends a key role in plant adaption or response in abiotic and biotic stress conditions (Iqbal et al., 2013). Ethylene production in plants frequently increases the tolerance level to sub-optimal environmental conditions. This part is specifically crucial from both agricultural and ecological point of views. It also plays important role in fruit ripening, inhibition of root growth, abscission, flowering induction and tuberization. The practical use of ethylene, which is difficult in a gaseous state, made great progress after the discovery of 2-chloroethane phosphoric acid. This product, when applied in a powder form, penetrates the tissue where it liberates ethylene.
1.2.5 The role of physical factors in plant tissue culture
1.2.5.1 Light
In vitro plant growth is regulated by several internal and external factors in which light is most important. The illumination system for in vitro culture should provide light in the spectral region that is involved in photosynthesis and photomorphogenic responses
(Bula et al., 1991; Seabrook, 2005). Tissue culture and growth rooms have long being using artificial light sources including high pressure sodium lamps, florescent lamps, incandescent lamps and metal halide lamps etc., among these florescent lamps have been the most popular in tissue culture rooms (Economou and Read, 1987).
40 Chapter 1 General Introduction
1.2.5.2 Temperature
Temperature has deep impact on the development and growth of the plant (Guy,
1999; Browse and Xin, 2001). The plants which are open to heat stress or chill stress, have reduced chlorophyll (Chl) biosynthesis because of down regulation of gene expression and protein abundance of many enzymes included in tetrapyrrole metabolism (Tewari and Tripathy, 1998; Mohanty et al., 2006). Impaired chloroplast development and chl biosynthesis directs to reduce photosynthesis outside the optimum temperature, causing extensive harm to plant production. Temperature provides both ‘inductive’ and ‘maintenance’ signals in development and can be considered as an environmental factor. It can activate growth and developmental processes like germination, seed dormancy release, and vernalization and in vitro rooting (Alderson et al., 1988).
1.2.5.3 Media preparation
The culture media should preferably be prepared in an equipped be portion, which should be structured in a way to allow ease and convenience in cleaning and minimizing the risks and possibilities of contamination. The tap water, distilled water and gas should be supplied. Suitable water deionization or sterilization systems are also significant (Ahloowali and Prakash, 2002). Some devices are needed for improved and better performance like, a freezer, refrigerator, pH meter, hot plate, stirrer; heater, electric balances with varying weighing ranges and in addition to glassware and chemicals also bunsen burner (Brown and Thorpe, 1984). It quite eminent now that the errors or mistakes which happen in the process of tissue culture most often begin from wrong or incorrect media preparation, hence best quality water, clean glassware, clean and pure chemicals with correct amount of media components should be aided.
41 Chapter 1 General Introduction
1.2.5.4 Media sterilization
The avoidance of contamination in tissue culture media is vital for the complete process of plant proliferation and facilitate in containing the spreading of plant parasites. The impurity and contamination of media can be restricted through applying acidification, antimicrobial agents, or by filtration through micro-porous filters (Levin and Tanny, 2002).
The media sterilization is frequently acquired through autoclaving at the temperature between the range from 115 – 1350C. The autoclaving benefits are that the process is simplified and fast, whereas the main shortcomings are that certain components may decompose and media pH changes which result in losing its usefulness. For example autoclaving mixtures of glucose, sucrose and fructose caused a reduction in the gelling capacity of agar and impacting pH of the culture medium by forming the furfural derivatives because of sucrose hydrolysis (Torres, 1989). Filtration by microporus filters (0.22- 0.45) is also applied for thermolabile organic constituents like vitamins, amino acids and growth regulators (Torres, 1989). In plant tissue culture media the additives of antimicrobial agents are rarely used.
1.2.5.5 pH pH establishes various key aspects of the structure and activity of biological macromolecules. pH is the negative logarithm of the concentration of hydrogen ions.
Nutrient medium pH ranges from 5.0 to 6.0 for appropriate in vitro growth of explant. pH higher than 7.0 and lower than 4.5 generally stops the growth and development.
The pH of the medium changes during autoclaving and usually falls by 0.3 to 0.5 units after autoclaving. If the pH falls appreciably during plant tissue culture (the medium becomes liquid), then a fresh medium should be prepared. One should know that a
42 Chapter 1 General Introduction starting pH of 6.0 could often fall to 5.5 or even lower during growth. pH greater than
6.0 gives a fairly hard medium and a pH lower 5.0 does not permit suitable gelling of the agar.
1.2.5.6 Solidifying agent
Agar, the most popular solidifying agent, is a seaweed derivative. Agar is added to the nutrient medium which acts as a supporting agent for plant cultures. There are many other gelling agents’ like phytagel, gellan gum and agarose (Prakash et al., 2002).
Agar has various benefits as compare to number of other gelling agents; when it is combined with water, it simply melts down in a temperature range of 60-1000C and it is solidifies at about 450C and creates a gel which is stable at all the possible incubation temperatures. Agar gels don’t react with the media constituents and are not digested by the plant enzymes and is normally applied in the media at concentrations in the range of 0.8-1.0%. In case if the culture is submerged in liquid media the shaking or rotating the flask in an orbital shaker can provide aeration. It is reported that as a solidifying agent agar can have an effect on the development and growth of in vitro cultures. Stoltz (1971) explained the effects of concentrations of agar on the growth of mature Iris embryos. Romberger and Tabor (1971) found the impact of agar on the shoot apical meristems of Picea. Maintaining the sterility is a very important factor in this method to protect plant material against bacterial and fungal contamination.
Hence it is important to optimize these aspects for plant species on individual basis. In vitro propagation results in the multiplication of most favorable genotypes. It is also used to produce plants with high concentrations of bioactive compounds, resilient against diseases and climatic changes. Plant tissue culture techniques are being
43 Chapter 1 General Introduction employed largely for mass production of elite plant varieties, which are otherwise difficult to propagate rapidly by traditional means.
1.2.6 Practical aspects of plant cell tissue and organ culture
The term plant tissue culture has construed as general label for a broad subject now a days. It encompasses all aspects of maintenance and cultivation of any plant material
(single cell, tissue and organ) in vitro. However, more recently this technology has been exploited in a more applied context. For the purpose of practical aspects, five areas are notable: protoplast cultures, callus cultures, anther cultures, cell suspensions, and meristem and organ cultures (Neumanna et al., 2009).
1.2.6.1 Callus Culture
Callus (pl. calli) is an actively-dividing non-organized mass of undifferentiated and differentiated cells developed in vitro from peripheral layers of wounded plant parts which proliferate from the mother cells, on a culture medium in the presence of growth regulators. Callus is rather an abnormal tissue having the potential to generate normal embryoids and roots and in turn it grows into plantlets. Explants from both mature and immature organs can be induced to form callus. However, explants with mitotically active cells (young, juvenile cells) are generally good for callus initiation Callus may be hard (due to lignification of cell walls) or brittle and sometimes soft.
The callus growth within a plant species is dependent on various factors such as the original position of the explant within the plant, and the growth conditions. Auxin, at a moderate to high concentration, is the primary hormone used to produce callus. In some species, a high concentration of auxin and a low concentration of cytokinin in the medium promote abundant cell proliferation with the formation of callus. Callus may be serially subcultured and grown for extended periods, but its composition and
44 Chapter 1 General Introduction structure may change with time as certain cells are favoured by the medium and come to dominate the culture. Physiological traits such as the production of natural products are more stable in callus cultures as compared to their respective suspensions (Deus-
Neumann and Zenk, 1984). Moreover, callus cultures are more suitable as stocks as they have longer culture periods.
1.2.6.2 Organogenesis
It is the development of adventitious organs or primordial (embyoid) from undifferentiated cell mass (callus) in tissue culture. Season of the year, donor conditions of the plant, the age and physiological condition of the parent plant contribute to the success of organogenesis in cell cultures. It is also managed normally by a balance between auxin and cytokinin in growth media. In 1957, Skoog and Miller were the first who established that high ratio of cytokinin to auxin speeded the development of shoots in tobacco callus whereas high auxin to cytokinin ratio encouraged root regeneration.
Caulogenesis is a type of organogenesis through which only adventitious shoot bud initiation occurs in the callus tissue. It is known as rhizogenesis when it is applicable for root. Anomalous structures when developed during organogenesis are called organoids. The localized meristematic cells on a callus which give rise to shoots and/or roots are called as meristemoids.
1.2.6.3 Cell Suspensions
A culture of suspension is established through shifting the comparatively crumbly part of the callus in liquid medium and is kept within the appropriate environments of agitation, aeration, temperature and light and other physical parameters
45 Chapter 1 General Introduction
(Chattopadhyay et al., 2002). Often, cell suspensions are made by mechanical shearing of callus material in a stirred liquid medium, provided by a shaker to supply the cells with sufficient oxygen (Morris and Fowler, 1981). Ideally all cells should be isolated in a cell suspension, however there are normally a high percentage of cells occurring as multicellular aggregates under practical conditions in these cell populations. A supplement of enzymes is able to break down the middle lamella connecting the cells in such clumps. These suspension cultures normally comprise of a large variety of cell types, and are less homogenous as compare to callus cultures.
Moreover, plant cell cultures provide a valuable plateform for the synthesis of high- value bioactive secondary metabolites, running in managed environment, free from climate and soil. Cell cultures not only define yield standard phytochemicals in huge quantities but remove the existence of indulging compounds that happen in the field- grown plants as well. (Lila, 2005).
The first commercial use plant cells cultivation on mass scale was performed in stirred tank reactors of 750 liter and 200 liter to create shikonin by cell culture of
Lithospermum erythrorhizon (Payne et al., 1987). Developments in the field of cell cultures for the making of medicinal compounds has enabled the manufacture of a huge variety of pharmaceuticals such as terpenoids, saponins, alkaloids, steroids, flavonoids, phenolics and amino acids (Yesil et al., 2010). Some of them are now obtainable commercially through the market such as paclitaxel and shikonin (Taxol).
Various recombinant proteins have been created in plant cell culture, like edible vaccines, antibodies, enzymes, cytokines and growth factors (Hellwig et al., 2004).
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1.2.7 Applications and future prospects
Plant Cell and tissue culture has momentous contributions in crops development and improvement having great upcoming potential (Kumar and Kumar, 1996). In current years the research in tissue culture and plant cell has massively increased throughout the world including the developing countries. Tissue culture technology is extensively used these days for the improvements in the field crops, development of forests, gardening, horticulture and plantation crops for increased in quality and quantity of agriculture and forestry production. Tissue culture technology is now used on commercial basis worldwide and has great contribution in increasing the production of high quality of planting material. Presently these techniques are applied basically for big scale production of exclusive planting material with coveted features. Lately the main focus has been on genetic transformation, especially aimed at:
Increase in the production of secondary metabolites.
Production of nematocidal compounds, alkaloids, pharmaceutics, as well as
some new compounds that are not found in the entire plant.
Regeneration of resilient in plants against pests, herbicides and diseases.
Upgrade of cultures in bio reactors.
Plants with dissimilar morphological qualities.
Transgenic plants in order to produce vaccines etc.
These advancements have made extensive effects in the progress of medicinal plants also (Bajaj, 1998). Micro-propagation is advantageous in expedite primary issue of new varieties as compare to proliferation by customary techniques, e.g pineapple
(Drew, 1980) & strawberry (Smith & Drew, 1990). Micro-propagation is also utilized for advancing germ-plasm to keep disease free stock in measured ecological conditions
47 Chapter 1 General Introduction
(Withers, 1989) as well as in the long term through cryopreservation (Kartha et al.,
1985). Micro-propagation has shown good results for both elite and selected plants, and benefited the horticulture, agriculture and forestry (Conger, 1981; Drew, 1997).
There is lot of interest present globally to develop and promote in vitro methodology that allow the multiplying and breeding of commercially semi-woody, viable woody, basic food, ornamental, medicinal and industrial plants. The species which are under the threat of extinction should be preferred in germplasm conservation (Conger, 1981;
Deberg and Zimmerman, 1990; Drew, 1997).
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1.3 Research Objectives
Medicinal plants are the most vital source of life saving drugs for majority of the world’s population. Conventional ways of producing pharmaceutical drugs are time consuming and expensive. Through the use of modern biotechnology, plants with specific chemical compositions can be propagated on mass scale and improved genetically for the extraction of active bulk pharmaceuticals. In vitro regeneration holds incredible potential for the production of superior quality and low cost plant- based medicine. Aloe vera products whether fresh gel, juice or formulated products are in use for health, medicinal and cosmetic purposes for many years. The medicinal and cosmetic value of Aloe germplasm depends on the quality and quantity of its value added constituents. The isolation of its bioactive compound “Aloe mannan” also known as Acemannan or Acetylated mannan, has drawn prime attention of many research workers. Chemical synthesis of Aloe mannan and other Aloe vera based beneficial products of pharmaceutical and medicinal value is difficult and expensive.
Besides variations in plant composition because of geographic location or differences due to gel extraction techniques and sample preparation methods have contributed to inconsistencies and discrepancies in the results attained from many studies in terms of the chemical composition and biological activities of A. vera leaf gel.
Our research proposal aims to produce high yielding cultivar from the selected Aloe specie through application of plant tissue culture techniques, for bioconversion and mass production of its bioactive compound Aloe mannan. This would ensure a steady and uniform round the year supply, independent of seasonal variation that affect availability of these in the plants under natural conditions. It will also provide high growth rate and cost effective production comparable to production in vivo. This
49 Chapter 1 General Introduction research will open up new vistas for commercial production and utilization of plant products in food, cosmetic and pharmaceutical industry.
In the present study, attempts were made to explore the potential of a highly efficient multiplication method based on callus induction and regeneration of A. barbadensis.
The effect of various factors such as light/dark conditions and various types and concentrations of plant growth regulators on the efficiency of the multiplication system was studied. In another experiment, the potential of cell suspension culture for biotransformation of Adrenosterone (1) was explored.
Conventionally propagated Aloe barbadensis plants and in vitro cultured plants were also evaluated for somaclonal variations by using RAPD and ISSR marker analysis.
50
CHAPTER 2
MICROPROPAGATION
51 Chapter 2 Micropropagationn
2.1 INTRODUCTION
2.1.1 Micropropagation of Aloe vera
Aloe vera is traditionally propagated by vegetative means due to wide spread male sterility. The most apparent weakness of traditional propagation is the slow rate of multiplication; 3 to 4 offshoots are produced by a sole plant each year. In addition, it needs specialized conditions in order to maintain disease-and virus-free propagation material. Hence with the fast growth of Aloe product industries, there is a high demand of leaves used as raw material which cannot be met through conventional breeding methods. Hence with the introduction of new cultivars and the limited supply of virus- free clones of existing cultivars, there is a need for a more economical, efficient and practical method of propagation using available plant material to meet commercial demand of superior quality plant material for its commercialized cultivation. For multiplication of desired plant species on large scale in vitro plant regeneration systems and plant tissue culture technology provides alternative production methods. in vitro propagation of Aloe vera has been done by many scientists like Sanchez et al.,
(1988), Natali et al., (1990), Meyer & Staden (1991), Roy & Sarkar (1991), Corneanu et al., (1994), Richwine et al., (1995), Abrie & Staden (2001), Chaudhuri & Mukandan
(2001). Sanchez et al., (1988) carried out micro-propagation from shoot meristem and found that in vitro culture of Aloe barbadensis is extremely hard for both plant regeneration and callus induction. A DNA microdensitometric study was carried out on various tissues of Aloe barbadensis and through in vitro culture of various explants.
Natali et al., (1990) reported vegetative meristems to generate a highly effective and rapid multiplication system. Shoot apices, micropropagated when were cultured on nutrient medium supplemented with Kinetin and 2,4-D, within a month. Natali et al.,
52 Chapter 2 Micropropagationn
(1990) also found that when explants were transferred in media with 6-BAP and 2,4-D, their morphogenetic ability was sustained. Aloes have been cultured in vitro with 2,4-D and cytokinins in the growth medium (Groenewald et al., 1975; Natali et al., 1990).
Meyer and Staden in 1991 reported that axillary bud growth and adventitious bud formation was acquired with decapitated shoot explants of Aloe barbadensis Mill.
Meyer and Staden in 1991 found that maximal budding and rooting of shoots was attained on a modified Murashige and Skoog medium, when IBA was added (Meyer &
Staden, 1991).
Aloe in vitro propagation has also been done due to their great ornamental and medicinal value (Vij et al., 1980). A very slight work has been done regarding the callus culture of Aloe species due to the difficulty in formation of primary cultures because of the emission of the phenolic substances by explant. Only one report is available on the development of callus and plant regeneration from seed calli of Aloe pretoriensis (Groenewald et al., 1975). Roy and Sarkar (1991) reported fast proliferation by shoot formation from calli of Aloe vera. From the new axillary shoots grown on underground rhizomatous stem they have generated the callus formation in stem segments. In 1991, Roy and Sarkar reported that in order to reduce the secretion of explant phenolic substances, polyvinylpyrrolidone was added and MS media supplemented with 2,4-D and Kinetin was found to successfully initiate callus cultures.
From axillary shoots on MS medium excluding growth regulators (the existence of which was discovered to prevent the 1st stage of development) the micro-propagation of Aloe vera was done by culturing fragments. At the 2nd stage of progress including the newly formed plantlets, the existence of a magnetic fluid in the culture medium enhanced secondary shoot production, plant development and rhizogenesis
(Corneanu et al., 1994). Richwine et al., (1995) described the effect of shoot cultures
53 Chapter 2 Micropropagationn
of Aloe, Gasteria and Haworthia species through undeveloped inflorescence. Shoots were adjusted on MS medium comprising zeatin and later kept on medium having
Zeatin and BA. A quick propagation protocol was set-up for the highly threatened A. polyphylla. In vitro seed was germinated on MS medium, both in the presence and absence of sucrose. In 2001, Abrie and Staden cultured plantlets on medium having benzyl adenine (BA) only, or a combination of BA and NAA. After initial difficulties with browning, the explants quickly formed axillary and adventitious buds (Abrie & Staden,
2001).
It is also described by Chaudhuri and Mukandan, (2001) that the concentrations of plant growth regulators, auxins and cytokinins play a vital role in multiple shoot formation of in vitro Aloe cultures. Highest multiplication of shoots was acquired on medium having BA + Adenine sulphate + IAA. Baksha in 2005 reported the “Micro- propagation of Aloe barbadensis Mill, through in vitro culture of shoot tip explants”. In this work MS media complemented with BAP (2.0 mg/L) and NAA (0.5 mg/L) gave largest number of multiple shoots (10 per explants), from shoot tip cultures. Top result of callus induction was developed in MS media with Kn 0.5 mg/L and 2,4-D 0.5 mg/L and Aloe shoot formation with 1 mg/L BAP and 0.5 mg/L NAA in the study conducted by Choudhary and Ray on in vitro culture of Aloe vera plants in 2011. Most recent research on Aloe barbadensis plant was conducted by Adelberg in 2012, in which he reported that meta-topolin (MT) generate the multiplication at the optimal concentration of 10 μM and BA produced the highest number of plantlets at 3.2 μM with optimal proliferation at approximately 6 μM. His study concluded that, plants cultured on liquid medium had the same multiplication rate as compared to plants cultured on agar, but were twice as large from liquid at the time of transfer to green house.
54 Chapter 2 Micropropagationn
2.1.1.1 Advantages of Micropropagation over Conventional
Propagation
Micropropagation is a rapid clonal multiplication, in which new varieties or
introductions can be bulked up to enable larger quantities to be released.
Accurate programming of plant propagation, as tissue culture is
independent of climatic changes.
Conservation of endangered plant species.
Production of novel and improved plant species by genetic engineering.
Production of secondary metabolites.
Facilitates plant exchange between countries when grown under aseptic
cultures.
Germplasm can be stored longer under in vitro conditions.
Tissue culture could be a novel source of variation which could increase
diversity and used in breeding programs.
Micro-shoot tip culture is the technique of choice to remove viruses and
other pathogens from various plant species. The micro shoot tip culture
technique has the benefit of regenerating a single plant from a single,
minuscule (approximately 0.5 mm) shoot. Several pathogens, including
viruses, are removed by this method.
55 Chapter 2 Micropropagationn
Micropropagation offers an efficient method for rapid clonal propagation and a tenfold increase of plant shoot in a test tube every six weeks is not uncommon. This means that production of millions of identical plants from a single shoot is a matter of months.
Thus Plant tissue culture is a low cost technique in which the reduction of cost is attained by improving process effectiveness and efficient use of resources. The main benefit of micro-propagation is the fast production of top quality, disease free, uniform and identical planting material.
2.1.1.2 Disadvantages of Micropropagation
Short comings of micropropagation include the fact that the disease free and healthy plants that are replenished are still prone to re-infection, if the concept of clean cultivation is not executed in proper and regulated manner. Tissue culture requires technical knowhow. Although it is considered that tissue culture is clonal propagating technique which gives true-to-type plants, but some methods and plants are especially prone to provide off types in large numbers.
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2.2 MATERIALS AND METHODS
The scientific research was conducted in the Plant Tissue Culture and Biotechnology
Wing of the H.E.J Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi. Collection of locally grown Aloe ecotypes was carried out from ecologically separate locations of Pakistan and were screened for high acemannan content. Then an elite Aloe barbadensis variety was selected as the mother plant for micropropagation and enhancement of the yield of its active ingredient.
Incubation of in vitro plant cultures was done in the growth rooms at 25C ± 2C under
16 hour light/dark photoperiod in which the light was provided by 30 watt, florescent tube lights (Philips). Laminar flow cabinet (Technico Scientific, Pakistan) was used for performing tissue culture procedures, using sterile equipment and efforts were made to prevent contamination of cultures. Cultures were maintained in glass jars
(Borosilicate) with plastic screw caps.
Experimental data was analyzed by two-way ANOVA program and Duncan’s Multiple
Range Test (DMRT) was used for the comparison of means at p=0.05. Analytical techniques such as TLC, HPLC, FT-IR, ¹H-NMR were used in the process of investigating the extracts for the separation, identification, purification and quantitation of the bioactive compound.
2.2.1 Preparation of Plant Growth Regulators—Stock
In various experiments, both types of growth regulators were used, in auxins, Indole-3
Butyric Acid (IBA), Indole-3-Acetic Acid (IAA), Naphthalene Acetic Acid (NAA) and
57 Chapter 2 Micropropagationn
2,4-dichlorophenoxyacetic acid (2,4-D) were used. The cytokinins, employed were
Kinetin (KIN) and Benzyl Aminopurine (BAP). Stock solutions of all the plant growth hormones were prepared at concentrations of 1 mg/ml using 1N concentration of
NaOH. Deionized water was used to make up the volume appropriately as required.
However, stock solution for 2,4-D was prepared by first dissolving it in 100% ethanol and adjusting the volume with deionized water (Table 2a). The storage temperatures of all the plant growth hormones was 40C and were used employing sterile micropipette tips as per requirements prior to autoclaving.
Table 2a: Preparation and Storage of Plant Growth Regulators
AUXINS CYTOKININS Growth Regulators IBA IAA NAA 2-4,D BAP KIN
Molecular 203.20 175.20 186.2 221.0 225.3 215.2 Weight EtOH/ EtOH/ 1N EtOH/ 1N 1N Solvent 1N 1N NaOH NaOH 1N NaOH NaOH NaOH NaOH EtOH/ Diluent Water Water Water Water Water 1N NaOH Powder 4°C 4°C 15°C 15°C 4°C 4°C Storage
Liquid Storage 4°C 4°C 4°C 4°C 4°C 4°C
Sterilization CA/F CA/F CA CA CA/F CA/F
CA = Autoclavable F = Filter Sterilization
2.2.2 Media Preparation
The basic salt mixtures employed in this study were of MS basal salt mixtures,
(Murashige and Skoog, 1962), as described in Table 2b. Sucrose was used as the sole carbon source and was added as a solid at concentration of 3% (w/v). The pH of
58 Chapter 2 Micropropagationn
the media were adjusted by adding either 0.1N NaOH or 0.1N HCl, as required to a final value in the range of 5.6-5.8 (Hanna Instruments, pH 211). Phytagel was added as a solid support at a concentration of 0.2%. 25 mL of media were dispensed into each glass jar secured with plastic screw caps and properly labeled. The media were sterilized by autoclaving at 15 p.s.i and 120°C for 15 min (GLSC-AA-195-0400,
Scientific Supplies, Faisalabad, Pakistan).
2.2.3 Isolation of Explants
Young axillary buds of 2.0-2.5 inches and leaves of healthy mother plant having high acemannan content were collected from the field of H.E.J. Research Institute, as an explant material.
2.2.4 Cleaning of Explants
The explants were washed extensively with tap water to clean any adhered dust and soil particles. The explants were kept for 10 min in 2% solution of Tween-20, followed by rinsing with autoclaved distilled water.
2.2.5 Sterilization of Explants
Laminar flow cabinets were used for conducting sterilization. Surface sterilization of the explants was done by immersing them in 0.1% aqueous solution of mercuric chloride for 10 minutes. Then they were washed several times with autoclaved distilled water to remove the traces of mercuric chloride. Each explant was treated separately to prevent chances of cross contamination.
2.2.6 Inoculation of Explants on Culture Initiation Medium
Inoculums of meristematic explants were finally prepared by trimming at the base carefully, so that only the meristematic tissues are isolated without damaging the
59 Chapter 2 Micropropagationn
apical and axillary meristem. Each explant was treated separately to avoid cross contamination, which can result in the loss of valuable cultures. The sterilized explants were transferred onto simple MS medium (full strength) for the establishment of A. barbadensis shoots cultures. After 20 days, the in vitro grown shoots were used for the callus induction experiment. Murashige and Skoog (MS) basal agar media (1962), complimented with different combinations and concentrations of plant growth hormones were used for callus induction and plant organogenesis. Adjustment of the media pH to 5.7 was done by 1 M NaOH and/or 1 M HCl and it was gelled with 8.0 gm/L w/v agar. 25 mL of media were then distributed into the glass-jars of 250 mL.
Then the media was autoclaved for sterilization, at 121 ºC for 15-20 minutes under 15 p.s.i.
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Table 2b: Basic ingredients of MS media employed in the micropropagation of
Aloe vera (Murashige & Skoog, 1962)
NUTRIENTS COMPOUND CONC. (mg/L)
Macro nutrients NH4NO3 1650
KNO3 1900
CaCl2.2H2O 440
KH2PO4 170
MgSO4.7H2O 370 Micro nutrients KI 0.830
H3BO3 6.200
MnSO4.4H2O 22.300
ZnSO4.7H2O 8.600
CuSO4.5H2O 0.025
CoCl2.6H2O 0.025
Na2MoO4.2H2O 0.250
Iron Source FeSO4.7H20 27.850
Na2EDTA.2H2O 37.250 Vitamins Nicotinic Acid 0.50 Pyridoxine HCl 0.50 Thiamine HCl 0.10 Inositol 100 Amino acid Glycine 2.00
2.2.7 Callus Induction
Each explant was excised aseptically into small sections of about 1 cm x 1 cm which were placed in culture jars containing MS basal agar medium, in which different concentrations (0.0-8.0 mg/L) and combinations of BAP (6-Benzyl amino purine) and kinetin (0-2 mg/L) with NAA (α-Naphthaleneacetic acid), IBA (Indole-3-butyric acid),
61 Chapter 2 Micropropagationn
and 2,4-D (2,4-Dichlorophenoxy acetic acid) were added. Half of the cultures were incubated at 22 ± 2º C in dark and the rest in illumination with a 16 hrs photoperiod at photon flux density of 75 µmol m-2 s-1, provided by white fluorescent tube lights. The cultures were incubated for duration of 4 weeks and then the explants were transferred on the same fresh nutrient media. Data on the callus induction with reference to their description was recorded after four weeks of incubation in both dark and light conditions.
2.2.8 Organogenesis
After 1 month of incubation, calli were excised into small pieces of about 1.0 gm FW
(Fresh Weight) and cultured on MS medium, in which different combination ratios of
BAP and kinetin (0-2 mg/L) with NAA, IBA, and 2,4-D (0-0.5 mg/L) were added, to investigate their potential for organogenesis. Incubation of half of the cultures was done at 22 ± 2 ºC in 16/8 hrs photoperiod at photon flux density of 75 µmol m-2 s1 and the other half were incubated under dark condition to investigate the effect of light on plant regeneration frequency. After shoots had developed from calli, their tips were excised and sub-cultured on the same regeneration media for multiplication.
Regenerated shoots were transferred to the medium containing different concentration of rooting hormones. MS medium enriched with indole butyric acid (IBA) and naphthalene acetic acid (NAA) were used ranging from 0.25 to 1.5 mg/L for this purpose and to select the best concentration of the hormone.
2.2.9 Acclimatization
Regenerated shoots with adequate number and length of roots were carefully separated from the incubation jars. Roots were washed carefully and gently in running tap water to eliminate traces of media and were transferred into perforated bags filled with garden sand. Plants were kept in green net sheds, where the light penetration
62 Chapter 2 Micropropagationn
was 50%, and humidity was maintained at around 65% - 75%. After two weeks, the plants were given a ‘nutrient enrichment’ 1/4th of normal recommended concentration, consisting of N:P:K at a ratio of 20:20:20. After 8 weeks the plants were transferred to the fields and watered regularly.
2.2.10 Statistical Analysis
Experimental data were recorded after 1 month and analyzed using two-way ANOVA program and the means were matched using Duncan’s Multiple Range Test at p=0.05.
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2.3 RESULTS AND DISCUSSION
Aloe barbadensis having high levels of acemannan was selected as an elite mother plant for in vitro mass propagation. Efficient system of plant regeneration was developed through organogenesis from callus cultures derived from stalk sections of
Aloe barbadensis. The effect of light conditions and different concentrations of auxins and cytokinins in MS medium on in vitro callus induction and multiple shoot regeneration was studied is summarized in Tables 3,4,5,6.
2.3.1 Callus Induction
The present study provides a method of indirect regeneration of Aloe barbadensis via callus. Cell suspension cultures have also been established and biotransformation studies have been conducted. In this regard, along with the yield, the texture of the callus is of great importance since it ensures the equal distribution of the plant cells in the medium. For the biotransformational studies, the callus should be friable which ultimately results in the establishment of a very effective suspension culture i.e. having an equal or uniform distribution of single plant cells or micro clumps of calli.
Maximum amount of callus (5.65 ± 1.9) was obtained under dark conditions on MS basal medium supplemented with 2 mg/L NAA. The callus obtained was friable in texture and off-white in color. MS basal medium supplemented with 8 mg/L also induced good amount (5.32 ± 1.92) of compact and off-white callus. The results for callus induction are summarized in Table (3 and 4) and Figure (2 and 3).
2.3.2 Effect of Light
The effect of light conditions was studied for the induction of callus in Aloe barbadensis. Previous results shows that callus induction in Aloe vera, requires dark conditions and the presence of both auxins and cytokinins (Kawai et al., 1993;
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Velcheva et al., 2005). Our results support the previous findings, as in our case the maximum percentage of friable callus induction and growth was also achieved under the dark conditions (Table 4 and, Fig. 2c and d). The retarded growth of callus was observed in light (Table 3 and Fig. 2a and b), because induced light augmented the production of phenolic compounds which obstruct the growth regulator activity
(George, 1993). The color of the callus produced in light (mostly brown) also indicates the production of phenolic compounds (Table 3). No anti-phenolics or antioxidants were examined to overcome the problem of phenolics just to avoid the complexity of the media formulation.
2.3.3 Effect of Hormones
The effect of various types of plant growth regulators was also evaluated for their potential to induce callus in Aloe barbadensis MS (Murashige and Skoog, 1962) medium having 2 mg/L of NAA (-naphthalene acetic acid) showed maximum callus formation (Table 4, and Figure 2c and d). The callus obtained was friable and off-white in color. MS medium, added with 8 mg/L of NAA, also induced a good, light brown colored callus in the light. Again our results deviate from the previous reports regarding the use of growth regulators. Kawai and his coworkers in 1993 reported that callus induction in another Aloe i.e. Aloe arborescens required the presence of both auxins and cytokinins but in our case the maximum percentage of friable callus induction and growth was achieved by only using the auxin i.e. NAA. During the present study it was also observed that the higher concentrations of NAA are not equally effective for the callus induction. Our results also showed that the combination of both auxin (NAA) and BAP (6-benzyl amino purine), a cytokinin, retarded the callus growth. This is because the presence of cytokinins results in the oxidation of
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anthracene compounds which causes browning and ultimately death of explants
(Natali et al., 1990).
Based on these observations, it can be postulated that for callus growth in Aloe vera only auxin (NAA) is sufficient. Calli induced in this system were used to establish the cell suspension cultures to study the growth curve and biotransformation ability of
Aloe barbadensis.
Table 3: Effect of BAP and NAA on callus formation in Aloe barbadensis after
30 days of incubation under iIlumination.
Fresh Media BAP NAA Description of callus Weighta codes (mg/L) (mg/L) (gm) Color Texture C 1 0.0 0.0 0.0 - -
C 2 0.0 2.0 4.3 ± 1.1 Off-white Compact
C 3 0.0 4.0 5.2 ± 1.7 White Compact
C 4 0.0 8.0 5.5 ± 2.1 Light brown Compact
C 5 1.0 0.0 0.0 - -
C 6 1.0 2.0 2.4 ± 0.78 Light green Loosely Compact
C 7 1.0 4.0 3.7 ± 1.64 Greenish Compact
C 8 1.0 8.0 4.7 ± 1.45 Greenish Brown Compact
C 9 1.5 0.0 3.5 ± 1.72 Green Compact
C 10 1.5 2.0 4.2 ± 2.0 Green Loosely compact
C 11 1.5 4.0 4.4 ± 2.1 Green Compact
C 12 1.5 8.0 5.4 ± 2.3 Greenish Brown Compact
C 13 2.0 0.0 0.0 - -
C14 2.0 2.0 3.7 ± 1.65 Greenish Brown Loosely Compact
Greenish off- C15 2.0 4.0 4.1 ± 1.87 Loosely compact white
C 16 2.0 8.0 4.3 ± 1.98 Dark green Compact
a sign represents mean fresh weight ± standard deviation.
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Table 4: Effect of BAP and NAA on callus formation from explants in Aloe barbadensis, after 30 days of incubation under dark conditions.
Media BAP NAA Fresh Weighta Description of callus
codes (mg/L) (mg/L) (gm) Color Texture
C 1 0.0 0.0 - - -
C 2 0.0 2.0 5.65 ± 1.90 Off-white Friable
C 3 0.0 4.0 5.23 ± 1.87 White Loosely compact
C 4 0.0 8.0 5.32 ± 1.92 Off-white Compact
C 5 1.0 0.0 2.11 ± 1.45 Light green Compact
C 6 1.0 2.0 3.12 ± 1.62 Off-white loosely Compact
C 7 1.0 4.0 3.87 ± 2.32 Light brown Compact
C 8 1.0 8.0 3.96 ± 2.02 Brown Compact
Greenish C 9 1.5 0.0 2.24 ± 1.57 Compact Brown
C 10 1.5 2.0 2.85 ± 1.69 Off-white Compact
C 11 1.5 4.0 3.36 ± 1.83 Light brown Loosely compact
C 12 1.5 8.0 4.21 ± 1.09 Brown Compact
C 13 2.0 0.0 2.01 ± 0.97 Greenish Friable
C14 2.0 2.0 2.32 ± 0.95 Greenish Compact
C15 2.0 4.0 2.38 ± 1.04 Light brown Compact
C 16 2.0 8.0 3.12 ± 1.65 Brown Compact
a sign represents mean fresh weight ± standard deviation.
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(a) (b)
(c) (d)
Figure 2: Callus formation from explants in Aloe barbadensis, (a) and (b) under illumination, (c) and (d) under dark condition
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2.3.4 Organogenesis
2.3.4.1 Shooting
Highest regeneration was observed when MS medium was supplemented with a combination of 1.0 mg/L BAP and 0.1 mg/L IBA (Table 5). On this media, shoot formation and elongation were enhanced and healthy plantlets measuring 2.475 inch in height, were obtained along with maximum number of multiple shoots i.e. 12.725
(Fig. 3a and b). BAP alone was unable to induce shoots but when it was used in combination with IBA even with smaller concentration it resulted in vigorous shoot growth as can be observed in Fig. 3a and b. At higher concentration of IBA number of shoots and height were considerable but not as inferred in case of IBA with 0.1 mg/L.
Number of multiple shoots and average number of leaves was comparable between
R2 and R5 media but they differ from each other on the basis of average height of plants. However, here an increase in concentrations of both cytokinin/auxin didn’t show exponential plantlet regeneration.
Organogenesis in Aloe barbadensis is proved to be controlled by light, as well as relative ratio of some plant growth hormones in the media. The effects of both these factors are discussed in separate sections below:
2.3.4.2 Effect of Light
Plantlet regeneration was achieved under both light and dark conditions. Proliferating shoot cultures were obtained on MS basal medium, providing highest shoot regeneration frequency (Table 5 and Fig. 3a). Cultures incubated under dark conditions also regenerated successfully, however exhibited poor regeneration
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frequency (Fig. 3b). The results of present study differ from the previous findings associated with Aloe barbadensis (Jayakrishna et al., 2011) in which regeneration only occurred in light. The early stage of callus induction seems to be sensitive to light, as light probably controls shoot regeneration by modifying cytokinin response
(George, 1993). After regeneration, all the plants were kept and maintained under the light condition (with the intensity and photoperiod mentioned above) and they multiplied efficiently.
2.3.4.3 Effect of Hormones
The growth regulators especially cytokinins are one of the most vital factors for shoot proliferation affecting the response (Zakia et al., 2013). The required concentration of each regulator varies significantly rendering the plant being cultured (George, 1993).
During the current study, regeneration occurred in the existence of both auxin (IBA) and cytokinin (BAP). In 2005 Velchera et al., also reported the efficient shooting response by addition of BA in culture media, while 2iPR exhibited moderate response and least initiation was achieved by addition of CPPU and Kin in culture media. This is in consistency with the results of previous investigations about Aloe vera, in which BA was always used in combination with other auxins for optimum induction of shoots
(Meyer and Staden, 1991; Zakia et al., 2013). The researchers also described the efficacy of BAP amongst the cytokinins tested for efficient propagation in Aloe vera
(Singh et al., 2009). However, according to some contradictory reports given previously by Natali and his coworkers in 1990 and Meyer and Staden in (1991),
Kinetin was employed for better shoot proliferation instead of BA in Aloe vera. The cause of this variation might be associated with the plant genotype.
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2.3.4.4 Rooting and Acclimatization
Rooting of the shoots regenerated using tissue culture technology is an important task to acquire for tissue cultured plantlets. The response of micro-shoots rooting is stated to be managed by the growth regulators in the medium, basal salt composition, genotype including the cultural conditions (Murashige, 1977; Bhojwani & Razdan
1992). In order to induce rooting auxin is a requisite for most of the species. Most commonly used among them for root induction are NAA and IBA (Bhojwani and
Razdan, 1992). In the present study IBA and NAA are both employed for this objective at different percentages usually alone and in some cases in combinations. Both of the auxins were utilized to select the best hormonal concentration for A. barbadensis rooting in vitro. The days to grow the rooting ranged from 14 to 19 and no more growth of the roots was observed after 25 days which suggested that minimum 14 days would be required to induce rooting in this specie using IBA and after 25 days, rooting medium has to be replaced with fresh one.
According to the data obtained (Table 6 and Fig. 4a and b), maximum average number of the roots was 8.0 with average length of the root of 6.38 cm at 1.0 mg/L of
IBA with MS medium and 95% of the shoots were rooted at this concentration in 14-20 days. This is the best datum in all using concentrations of the hormones recruited. At other concentration of the IBA no considerable number and length of roots were observed. At the same concentration of IBA, NAA did not respond as same as IBA because average no. of root was 4.8 and average length of roots was 5.26 cm, although the percent of rooted shoots was 90% and it took almost similar time period as IBA. Previously many researchers reported in vitro rooting in Aloe vera by using
IBA (Sanchez et al., 1988; Meyer and Staden, 1991; Richwine et al., 1995).
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In the current study the MS medium added with NAA only or combined with BAP proved to be inefficient for rooting of regenerated plants. The regenerated plantlets acclimatized well in green house conditions. For hardening of Aloe vera a mixture of light soil and sand (1:1) or soil, sand vermiculite or perlite has been recommended by researchers (Natali et al., 1990). The regenerated plants were phenotypically identical to the mother plants.
Table 5: Effects of BAP and IBA on plantlet regeneration from calli in Aloe barbadensis.
Number of Height of Media Number of BAP (mg/L) IBA (mg/L) multiple plants codes leaves shootsa (inch)
R 1 1.0 0.0 0 efg 0 efg 0 efg
R 2 1.0 0.1 12.725 a 1.75 a 2.475 a
R 3 1.0 0.5 10.95 abc 1.45 abc 1.15 c
R 4 2.0 0.0 0 e 0 e 0 e
R 5 2.0 0.1 12.5 ab 1.5 ab 1.475 ab
R 6 2.0 0.5 11.625 bcd 1.15 bcd 1.15 cd
Means with the same letters are not significantly different at p=0.05 (DMRT).
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(a) (b) Figure 3: Plantlet regeneration from calli in Aloe barbadensis, (a), (under illumination and (b) in dark conditions.
Table 6: Effect of Auxins on Root formation from Regenerated Plantlets
Hormone Concentration No. of rootsa Length of % of rooted Rooting root (cm) plantlet period (days) IBA 0.25 1.8 ± 0.83 3.53 ± 0.33 60 17-22
0.5 2.4 ± 1.10 4.35 ± 0.26 70 15-20
0.75 3.4 ± 0.54 5.43 ± 0.14 85 15-20
1.0 8.0 ± 0.70 6.38 ± 0.34 95 14-20
1.5 3.2 ± 0.83 5.34 ± 0.17 75 20-25
NAA 0.25 2.2 ± 0.83 3.54 ± 0.61 60 15-21
0.5 2.8 ± 1.48 3.32 ± 0.15 70 18-24
0.75 3.0 ± 0.70 4.46 ± 0.09 80 16-22
1.0 3.8 ± 0.44 5.26 ± 0.18 90 15-20
1.5 3.2 ± 0.83 4.82 ± 0.83 85 19-24
aeach treatment is comprised of 5 replicate and is given in mean ± Standard deviation
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(a) (b)
Figure 4: Formation of roots in regenerated Aloe barbadensis, (a) and (b)
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CHAPTER 3
QUALITATIVE AND QUANTITATIVE ANALYSIS
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3.1 INTRODUCTION
3.1.1 Qualitative and Quantitative Analysis of Acemannan
Aloe vera leaves are made up of a thick epidermal layer which surrounds the mesophyll tissues, which are differentiated into thin-walled parenchyma cells and collenchyma cells. The inner parenchyma is composed of parenchymatous tissues which comprises of 0.5% solid matter and 99% water. In 1999, Femina and co- workers observed that polysaccharides constitute the major portion of the Aloe vera parenchyma having two main kinds of polymers: a storage polysaccharide
“acemannan” was located in the protoplasts of cells and a broad range of polysaccharides formed matrix of the cell wall. Aloe vera gel is obtained from the leaf by the process of filleting then subjected to homogenization, alcohol extraction to yield a complex high molecular weight carbohydrate. In the early 1980’s, an acetylated polymannose, acemannan, was identified as an active component of Aloe vera gel
(Johnson et al., 1989). This carbohydrate was commercially designated as acemannan by the United States (US. Patent WO/1993/008810). According to the literature reports, significant seasonal and cultivar variations affect the quantities of mannose residues in the storage polysaccharide within the parenchymal cells, leading to huge fluctuations in the polysaccharide composition of A. vera fillet (Femenia et al.,
1999).
A wide range of in vivo and in vitro immunostimulatory activities of polysaccharides isolated from Aloe vera plant gel have been documented (Miles et al., 2002;
Kardosava, 2006). Acemannan is considered to be the key biologically active substance found in the Aloe vera plant gel (Debra et al., 1988; Reynolds & Dweck,
1999; Lee et al., 2001). Acemannan, a β-(1, 4)-linked polydispersed, highly acetylated
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mannan has an average molecular weight of 1000 kDa (Turner et al., 2004; Tai-Nin
Chow et al., 2005). The quality of Aloe vera gel preparations can be accessed by 1H
NMR technique, which has proven to be an essential tool for observing the presence of the bioactive polysaccharide acemannan. According to Manna & McAnalley (1993), acemannan is a linear polysaccharide consist of β-(1→4)-linked mannan partly acetylated in positions 2, 3 or 6, having galactose units attached to C6 forming some side chains (Manna & McAnalley, 1993; McAnalley, 1993). In H-NMR spectrum, the multiple peaks of acetyl groups produce a characteristic signal from 2.0 to 2.3 ppm, which can be reflected as the benchmark of Aloe vera (Bozzi et al., 2007). Structure of
Acemannan monomer is given below:
Figure 5: Structure of polydispersed β-1, 4-linked mannan substituted with O-acetyl groups (acetylated mannan; aloe polysaccharide/acemannan), (Prerak Patel et al.,
2012).
3.1.2 Acemannan Isolation and Purification Studies
Modern science in the early 20th century was applied in order to recognize and identify the active ingredients in the design cultivation of Aloe vera, processing and harvesting techniques to store, preserve and familiarize Aloe vera into larger commercial markets. The Aloe active ingredient acemannan having immense
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therapeutic potential and applications has been subjected to various, isolation, separation and purification studies (Acemannan Review by Kenneth and Yates, 2012).
Assays of acemannan were developed and purified through various chromatographic techniques including HPLC (Okamura et al., 1996, Talmadge et al., 2004; Monge et al., 2008), gel permeation chromatography (McAnalley, 1993; Femenia et al., 1999,
Femenia et al., 2003; Choche, et al., 2014) and size exclusion chromatography
(Turner et al., 2004; Alonso et al., 2012).
Earlier studies have shown that acemannan is not steady under various conditions utilized in the manufacturing process including acid, heat, enzymes discharged from cells while processing (Femenia and Garcia, 2003). Unfortunately due to improper manufacturing processes used by many Aloe product manufacturers, acemannan is partially and sometimes fully removed, having Aloe products with little or no acemannan (Turner et al., 2004). Moreover, currently most manufactures do not assay for acemannan content in their final products. Instead the presence of adulterants, impurities, and preservatives have been found in many leading Aloe based commercial products (Bozzi et al., 2007). The varying and inconsistent quality of Aloe vera preparations have resulted in numerous conflicting scientific studies and introduction of several products into the market place of variable quality causing conflicting consumer experiences, and collectively emerging in controversy about the true medicinal benefits of Aloe vera.
Thus designing operationally viable and scalable productive processes to obtain bioactive acemannan for successful clinical trials has been a challenge for Aloe research and development laboratories. These processes were analyzed on the basis of several parameters including recovery, operation time for each step, physical-
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chemical characteristics, purity, bioactivity and consumer safety of the acquired product.
3.1.3 Chromatography
Chromatography is an extremely effectual and powerful analytical technique for the separation, accurate identification and quantitative ascertaining of compounds in a complex mixture. The mixture is dissolved in a liquid known as mobile phase, which carries it through an inert solid support called the stationary phase. In this technique separation of the components is based on their retention ratios or affinity to these phases. The stationary phase can be a liquid or solid, supported by solid support of chemical and physical forces, while mobile phase may be gaseous or a liquid.
Chromatography can be preparative or analytical. Preparative chromatography is applied to disintegrate and purify the components of mixture, whereas by analytical chromatography the relative proportions of analytes or the quantity in a mixture can be precisely measured and needs comparatively lesser amounts of material.
The chromatographic methods include, thin layer chromatography (TLC), paper chromatography (PC), high performance liquid chromatography (HPLC) and gas chromatography (GC). Recently, ultra performance liquid chromatography (UPLC) has been established and commercialized.
3.1.4 A Brief History
The meaning of the term chromatography is “color writing”, was founded by a Russian botanist named Mikhail Tswett, who in 1903 split the plant pigments on chalk packed in glass columns (Ettre, 2002). The chemists use to apply gravity fed silica columns for purifying organic materials and ion-exchange resin columns to split the ionic compounds and radionuclides since 1930. In 1952 the successful application of gas
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chromatography (GC) by British chemists A.J.P Martin and co-workers, has provided the theoretical foundation as well as the encouragement for the development of Liquid chromatography (LC) in which physical parting method was applied in the liquid phase. In the late sixties with the use of small-particle columns requiring high pressure pumps, LC turned “high performance”. The researchers of 1960s including Hovath,
Kirkland and Huber, developed the first generation of high-performance liquid chromatographs. HPLC became a sensitive quantitative method due to the commercial development of in-line detectors and reliable injectors leading to a huge growth of applications. Later in 1980s, HPLC has become essential in pharmaceuticals and other industries due to its accuracy and flexibility. During recent years, due to the increasing needs of life sciences and pharmaceutical industry HPLC is continuously improving fast towards increased efficiency, higher speed and sensitivity.
3.1.5 High Performance Liquid chromatography (HPLC)
HPLC is a physical splitting methodology where a sample is broken into the fundamental components or analytes by injecting small quantity of liquid sample in a packed tube (column) filled with small particles (3 to 5 micron μm) in diameter known as the stationary phase, in which discrete components of the sample are mixed with a liquid or eluent (mobile phase) pressurized through the column by a high force delivery pump. Through the column packing these components are detached from each other that include many chemical and/or physical interactions among packing particles and their molecules. These separated components also called as “eluate” are identified at the end of the tube (column) by a flow-through device (detector) through which their volume is measured. An output from this detector is known as
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“liquid chromatogram”. Principally, LC and HPLC work similarly except that the sensitivity, efficiency, speed, and ease of operation of HPLC are much better.
The most important part of the system in the HPLC column is the stationary phase where parting occurs. Various forms of analysis are categorized and built on the nature of stationary phase and mechanism after the segregation in the column.
There are normally three types of interactions:
Polar Interactions
Variances in polarity among the sample components and the bonding entities on stationary phase results in preferential retention
Ionic Interactions
Separation founded on charge properties of sample molecules. Analytic ions have attraction for oppositely charged ionic centers on the stationary phase
Molecular Size
Separation occurs because of entrapment of little molecules in the stationary phase pores. Big molecules pass through first and succeed by elution of smaller trapped molecules. Eluent polarity performs the top role in all types of HPLC. There are two elution types: isocratic and gradient. In the first type constant eluent composition is pumped through the column throughout the entire analysis. In the second type, eluent composition (and strength) is gradually transformed throughout the run. Column length with the similar stationary phase has important influence on separation. Long and wide columns can take greater sample loads and give greater resolution. On the other
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side shorter columns cut analysis time which results in lower mobile phase consumption.
High performance liquid chromatography (HPLC) is a multipurpose analytical technology broadly applied for the analysis of pharmaceuticals, biomolecules, polymers, and many organic and inorganic compounds.
3.1.5.1 Types of HPLC
HPLC can be classified into four types based on nature of the stationary phase and major separation modes.
1. Normal phase and adsorption chromatography
2. Reversed phase chromatography
3. Ion exchange chromatography
4. Size exclusion chromatography
3.1.5.1.1 Normal Phase or Adsorption Chromatography
In this type of chromatography the stationary phase consists of polar material such as silica, cyanopropyl-bonded, amino-bonded, etc. while mobile phase is non-polar in nature like hexane, iso-octane, methylene chloride, ethyl acetate. The polar compounds are retained more by the polar stationary phase resulting in the early elution of non-polar compounds. Normal phase separations are completed less than
10% of the time. The method is beneficial for water-sensitive compounds, cis-trans isomers, geometric isomers, chiral compounds and class separations.
3.1.5.1.2 Reverse Phase Liquid Chromatography (RPLC)
RPC is, by far, the most popular mode, used by over 90% of chromatographers. In this type of chromatography, the stationary phase is non-polar (Hydrocarbons e.g.
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C18, C8, C3, phenyl, etc) while mobile phase is polar (e.g. methanol, acetonitrile or water) in nature. Here non-polar compounds face longer retention in the column and polar compounds elute first. The method can be used for polar, non-polar, ionizable and ionic molecules making RPC very versatile.
3.1.5.1.3 Ion Exchange Chromatography
In ion exchange chromatography the column packing of the stationary bed contains ionic groups of opposite charge to the sample ions (e.g. sulfonic, tetraalkylammonium). The mobile phase is an aqueous buffer e.g. phosphate, formate, etc, where both pH and ionic power are applied to regulate elution time. This method is applied almost absolutely with ionic or ionizable samples. The powerful the charge on the sample, the firmer it will be attracted to the ionic surface and therefore, the lengthier it will take to elute. Ion exchange is applied around 20% of the liquid chromatographers. The method is suitable for parting the organic and inorganic cations and anions in aqueous solution. Amino acids, ionic dye, and proteins can be detached by ion exchange chromatography.
3.1.5.1.4 Size Exclusion Chromatography (SEC)
In SEC separation occurs on the basis of molecular size of molecules and there is no interaction among the column packing material and the sample compounds. Instead, molecules diffuse into pores of a porous medium and must easily enter and exit pores to be detached. Molecules are detached on the basis of their size relative to the pore size. Large molecules elute first, succeed by intermediate size of molecules and ultimately the smallest size molecules elute in the end. The SEC method is applied by
10 to 15% of chromatographers, mostly for protein and polymer characterization.
There are two modes:
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Aqueous SEC sometimes referred to as Gel Filtration Chromatography (GFC).
Non-aqueous SEC sometimes termed Gel Permeation Chromatography (GPC).
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3.2 MATERIALS AND METHODS
3.2.1 Selection of Mother Plant
The Aloe vera species were collected from various cities of Sindh, Punjab,
Baluchistan and Khyber Pakhtunkhwa and planted in the field of H.E.J. Research
Institute of Chemistry, University of Karachi. Screening of the plants was done to identify disease free and healthy specimens. Best mother plants were selected on the basis of high yield of leaf biomass and were evaluated physically for the absence of any contamination. Plants are decontaminated by spraying them with agents such as pesticides, fungicides or insecticides. Preferred fungicides for the pretreatment of the mother plant include Benlate and Topsin M at a concentration of about 0.05% to 0.2%.
The plants were tested for high acemannan content using standard analytical techniques including TLC, HPLC, FT-IR spectroscopy and ¹H-NMR spectroscopy. The identification of known constituents was done by comparing their acquired and reported spectrums.
3.2.2 Acemannan Isolation and Purification
3.2.2.1 Plant collection for acemannan analysis
Four different varieties of Aloe vera were collected from Karachi, Islamabad, Quetta and Hyderabad. Botanical identification was done by the Botany Department,
University of Karachi. Their names with their designated codes are: Aloe alfredii (A1),
Aloe barbadensis (A2), Aloe dwetii (A3), Aloe bellatula (A4). From 2 kg leaves of each variety 30 mg of gel extract was subjected to various extraction, isolation and purification steps to obtain acetyl mannan dry powder.
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3.2.2.2 Isolation and Purification
Aloe vera gel extracts of 2 kg leaves of different Aloe varieties were provided by collaboration of P.C.S.I.R. laboratories, Karachi, Pakistan. The gel samples were kept at 4ºC for 2 hrs. Acetylated mannans in the gel of polysaccharides were extracted and isolated by alcohol precipitation method (McAnalley, 1993), with some modification.
The clear inner gel was homogenized and most of the pulp removed. The viscous gel was brought to an acidic pH of approximately 3.20 with dilute HCl, which solubilizes the oxalates and lactates of calcium and magnesium that are normally present. Then extraction of the gel was done for 5-6 hours with 4 volumes of 95% ethanol at room temperature and kept overnight at 40C for maximum precipitation of the bioactive polysaccharide. The fibrous floating particles were removed and the water/alcohol mixture was discarded. Then centrifugation (Hareus, Biofuge) was done at 10,000×g for 30 min at 40C to obtain the solid precipitate. Most substances such as oligosaccharides, organic acids, monosugars, inorganic salts and anthraquinones which are soluble in the water/alcohol mixture were eliminated in the process. The deposit was then dissolved in deionized water and passed through 0.22 µm filters
(Millipore), using high pressure vaccum pump filteration (Millipore), lyophilized (Freeze
Dryer Trio Science, Model TR-FD-BT-50) and stored at -200C until further analysis.
3.2.3 Characterization and Qualitative Analysis
Acetylated polymannose, was characterized using TLC, HPLC, FT-IR spectroscopy and ¹H-NMR analysis.
3.2.3.1 Fourier Transform Infrared (FT‐IR) Spectroscopic Analysis
The functional groups of acemannan absorb at characteristic infrared frequencies.
Infrared spectroscopy therefore is an important method to characterize this material.
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3.2.3.1.1 Chemicals
Infrared grade potassium bromide (KBr) powder (Merk).
3.2.3.1.2 Instrumentation
Broker Fourier Transform infrared (Ft-Ir-8900) spectrophotometer model Vector-22 equipped with a Pentium 2 Computer and a HP 640 C Deskjet printer/plotter.
3.2.3.1.3 Sample Preparation
Acemannan was pre-ground to fine powder by using Wiley Mill (Thomas Scientific
CO.) and a screen which allows particles smaller than 60 mesh size. 1mg pre-ground sample was mixed with 30 mg of dry KBr powder. The mixture was reground by hand using agate mortar and pestle to a homogeneous material. Then the sample was pressed into a transparent disc using a hydraulic jack (Riken Power) at a pressure of
40,000 psi. The disc was scanned from 4000 cm ¹ to 400 cm. a multiple scan (20 scans) was performed with a 2 cm resolution to improve signal to noise ratio.
3.2.4 Nuclear Magnetic Resonance Spectroscopy (NMR)
Nuclear magnetic resonance spectra were recorded in D2O on Bruker AV 500 NMR spectrometer using the Windows top spin 2.1 operating system, at 500 MHz, for ¹H-
NMR.
3.2.5 Acemannan determination from Aloe vera calli under carbohydrate stress conditions.
Aloe plant calli were subjected to different carbohydrate stress conditions in order to study the effects of various concentrations of carbohydrates on acemannan production and enhancement. Calli were sub-cultured on MS media having Sucrose
(2-6%), Mannose (2-6%), Glucose (2-6%) and Galactose (2-6%). Each treatment had
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5 replicates. After 30 days of incubation in dark conditions calli were pooled, harvested and fresh weight was calculated. The callus crude extract was obtained by the method described by McAnalley, 1990 with some modifications. Briefly, each callus sample was ground to powder by liquid nitrogen. The samples were dissolved in water and homogenized to remove most of the pulp. The pH of the callus sample extracts was adjusted to 3.20 with dilute HCl. Then extraction was done for 4-5 hours with four volumes of 95% ethanol at room temperature and kept overnight at 4 ºC for maximum precipitation of the bioactive polysaccharide. This was followed by centrifugation (Hareus, Biofuge) at 10,000 × g for 30 min at 4 ºC. The supernatant was siphoned off and deposit was dissolved in deionized water and passed through 0.22
µm filters (Millipore), using high pressure vaccum pump filteration (Millipore), lyophilized (Freeze Dryer Trio Science, Model TR-FD-BT-50) and stored at -20 ºC until further analysis.
3.2.6 TLC Analysis
The methanolic extracts were studied and analyzed by reverse phase thin layer chromatography (RP-TLC) using RP-18 silica gel coated glass plates by co- chromatography with acemannan standard. The plates were developed in a solvent system of methanol: water (2:8), dried and detected using ceric sulphate spray reagent.
3.2.7 Recycling Preparative High Performance Liquid Chromatography
3.2.7.1 Reagents and Chemicals
Methanol and acetonitrile (HPLC grades) were purchased from Fisher Scientific. AMP
Floracel ®, USA, containing 90 mg powder of Aloe mucilaginous polysaccharides
(AMP) extracted from Aloe vera plant was used to obtain Aloe vera mannan used as
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reference standard. Commercially available Aloe vera samples do not have acemannan in purified form. Thus an isolation and purification method of this bioactive compound was developed and optimized by using Recycling Preparative High
Performance Liquid Chromatography HPLC Waters Alliance (ODS-M-80). The mobile phase was MeOH:H2O (20:80) with flow rate 3 mL/min, to obtain acemannan (8; 10 mg; retention time 40 min). Estimation of acemannan was done by comparing spectras of FT-IR and ¹H-NMR with the literature reports.
3.2.7.2 Chromatographic Conditions
The dried crude extracts were dissolved in solvent MeOH:H2O with the ratio of (20:80) and extraction of samples was done by sonication (Sonicator from Fisher Scientific) for 10 min. Samples were vortexed by using Vortex Mixer (Fisher Scientific). Then the extract was filtered with a 0.45 µm syringe filter, prior to HPLC analysis. The samples were further purified by preparative recycling HPLC (Waters Alliance, (ODS-M-80) and detector set at wavelength of 210 nm. The mobile phase was MeOH:H2O (20:80) with flow rate of 3 mL/min to obtain acemannan (8; 10 mg; retention time 40 min).
3.2.8 Quantitative Analysis
3.2.8.1 Instrumentation
The HPLC analysis of standard acemannan and sample extracts was carried out on a
Waters Alliance 2695 HPLC system with automated gradient controller, 2996 photodiode array detector connected to a computerized data station using Waters
Empower Software Build 1154.
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3.2.8.2 Sample Preparation
Sample preparation is a very important step in the development of standard analytical procedures required for the analysis and standardization of herbal and botanical preparations, which must be effective and reproducible. The protocol for the extraction of Aloe vera samples was optimized and different parameters such as solvent systems, HPLC columns, time and temperature were carefully studied in order to validate the efficiency of the extraction method.
For quantitative analysis, dried plant extracts were dissolved in MeOH:H2O (20:80) and extraction of samples was done by sonication (Sonicator from Fisher Scientific).
Samples were vortexed by using Vortex Mixer (Fisher Scientific). Then the extract was filtered with a 0.45 µm syringe filter, prior to HPLC analysis. The mobile phase (80% water and 20% methanol) was sonicated for 20 min for removing air bubbles prior introduction to HPLC system.
3.2.8.3 Chromatographic Conditions
Chromatographic conditions were optimized to obtain the best separation of standard peak, in the standard mixture as well in the Aloe vera extracts. Stationary phase used was the reverse phase C-18 (250 × 4.6 mm) and the column temperature was 37 ºC.
The mobile phase consisted of 80% water and 20% methanol. Analysis was carried out by isocratic elution at a flow rate of 1.0 mL/min, with injection volume of 20 µL. The total analysis run time of each sample was 10 min, which was followed by a 5 min wash with 100% methanol. The peak of acemannan standard and all the samples were detected at a wavelength of 210 nm. For correct identification of the peaks the comparison of the UV spectra and retention times was done with standard compound.
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3.2.9 Preparation of Standard Calibration Curve
Standard stock solution was made by diffusing 10 mg of purified acemannan standard in 10 mL of 20% methanol and 80% water to make solution. Then the solution was vortexed and filtered by using 1 mL syringe and 0.45 micron filter assembly. Then different concentrations in the range of 0.1-10 mg/10 mL were prepared by serial dilution for HPLC-UV analysis. The peak areas of each concentration were recorded and the standard calibration was constructed by plotting the concentration on X-axis and corresponding peak area was plotted on the Y-axis.
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3.3 RESULTS AND DISCUSSION
Plant tissue culture is a promising technology, especially for the multiplication and production of novel and improved plants species and for an increased biosynthesis of products of industrial and medicinal value from vegetative resource. It is becoming popular as a commercial methodology for mass-scale propagation of the elite varieties, especially for plants which are complicated or difficult to propagate fast by traditional techniques. Micropropagation is the process of in vitro regeneration of plants through organs, cells, tissues or protoplast applying methods like tissue culture for producing true-to-type resultant plants of a selected genotype. Generally, tissue from a plant commonly known as an explant is isolated from a plant whose propagation is required to create a sterile tissue.
In the present study an elite variety of Aloe barbadensis, having high acemannan content was investigated and exploited for micropropagation and enhanced production of its bioactive compound acemannan. Attempts were made to explore the potential of a highly efficient multiplication method based on callus induction and regeneration of
A. barbadensis. The effect of various factors such as light/dark conditions, various types and concentrations of plant growth regulators and carbohydrate sources, on the efficiency of the multiplication system as well as on acemannan production and enhancement was evaluated.
3.3.1 Selection of Elite Mother Plant
Four different varieties of Aloe vera were collected from different cities of Pakistan.
From 2 Kg leaves of each variety 30 mg of gel extract was subject to various isolation and purification steps to obtain acetyl mannan dry powder. The concentration of
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acemannan in four different varieties was studied which demonstrate the varied specie production of this anti-cancer molecule having numerous pivotal biological activities. All varieties were taken from field as described in experimental section.
From all the varieties tested, Aloe barbadensis (A2) had maximum quantity of acemannan i.e. 0.43 mg from 10 mg of the gel extract analyzed, whereas minimum concentration was in A. alfredii, which was 0.05 mg as represented in Graph 1.
Analysis of acemannan concentration in Aloe 12 vera varieties 5 4.30 10 4
(mg) 10.00 10.00 10.00 10.00 8 gel 3
vera 6
Acemannan (mg/ml) 2.10 2 of
Aloe 4 1.30 of 1 2 0.50
Weight 0 0 Aloe vera gel (A1)Aloe vera gel (A2)Aloe vera gel (A3)Aloe vera gel (A4) Concentration Weight of gel sample(mg) Concentration of acemannan (mg/10 mL)
Graph 1: Shows the analysis of acemannan concentration in different Aloe varieties
3.3.2 FT‐IR Spectrum Analysis
The IR spectra summarized clearly indicates the presence of the bioactive polysaccharide acemannan in the Aloe varieties (Table 7 and Figure 7) and in Aloe barbadensis callus cultures and in vitro regenerated plants from callus (Table 8), which was referred against standard A, (Acemannan Immunostimulant (AMP Floracel
®, USA), Figure 6.
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Figure 6: FT-IR spectrum of Aloe vera standard
Figure 7: FT-IR spectrum of Aloe vera plant gel
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Table 7: Shows the wavenumber (cmֿ¹) of acemannan in Aloe species
Wavenumber (cm-¹) A A 1 A 2 A 3 A 4 C-O 1068.3 1077.7 1078.1 1091.6 1090.2 (Pyranose ring) O-H 3429.9 3415.7 3428.7 3364.3 3409.6 C-O-C 1245.9 1272.1 1270.4 1242.3 1240.0
C 1737.8 1724.7 1726.7 1720.0 1700.0 (Carbonyl) C-H 1376.2 1304.6 1386.8 1323.9 1320.0 (Bending) C 1635.5 1663.4 1620.8 1645.5 1662.2 (Acetyl) C-H 2923.3 2924.2 2928.5 2928.6 2925.2 (Stretch)
Table 8: Shows the wavenumber (cmֿ¹) of acemannan in callus cultures and in
vitro regenerated Aloe barbadensis plants from callus
(Wavenumber (cmֿ¹ A C7A (D) C7A(L) C7A* (D) C7A* (L) C-O 1068.3 1076.2 1078.1 1039.6 1076.2 (Pyranose ring) O-H 3429.9 3452.3 3446.6 3413.8 3456.2 C-O-C 1245.9 1272.9 1272.9 1250.0 1276.8
C 1737.8 1700.0 1700.0 1700.0 1700.0 (Carbonyl) C-H 1376.2 1394.4 1305.7 1300.0 1396.4 (Bending) C 1635.5 1600.0 1600.0 1645.2 1600.0 (Acetyl) C-H 2923.3 2923.9 2923.9 2923.9 2925.8 (Stretch)
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Note: * C7A represents callus cultures in MS media supplemented with 2 mg/L NAA and 3% sucrose for. C7A* indicates in-vitro plants regenerated in MS medium supplemented with a combination of 1.0 mg/L BAP and 0.1 mg/L IBA. Dark and light conditions are represented by (D) and (L) respectively.
Brief Description of Functional groups:
C--O stretch of pyranose ring structure, (ketone group). O--H stretch, (hydroxyl group). C--O--C stretch, (acetyl group). C= stretch (carbonyl group). C--H bending. C= amide carbonyl stretch, (Amide I). C--H stretch.
The spectrum of bioactive polysaccharide acemannan (A) demonstrates a strong
The carbonyl and C--O--C .¹־absorption due to O--H stretching about 3429 cm
respectively. The ¹־stretches of acetyl group are located near 1737 and 1245 cm strong single band of C--O--C system is indicative of O-acetyl group bonded equatorially to the monomer unit. Thus, FT-IR spectra mentioned above clearly indicates the presence of this bioactive compound (Krokida, et al., 2011; Prerak Patel et al., 2012; Ray, et al., 2013).
3.3.3 Determination of Acemannan in conventionally propagated, in vitro regenerated Aloe vera plants and calli
In the present study the genetic variability of regenerated plants and conventionally grown Aloe barbadensis was investigated and exploited for the enhanced production
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of bioactive acemannan. The maximum concentration of acemannan as summarized in Table 9 is observed in the in vitro regenerated Aloe barbadensis plants cultured in media having 3% sucrose under dark conditions i.e 6.1 mg/10 mL as compared to the soil grown and tissue cultured plants and callus cultures under light conditions.
Table 9: Shows analysis of Acemannan concentration in conventionally propagated Aloe barbadensis plant, in vitro callus cultures and regenerated plant from calli under both light and dark conditions.
Media Weight of Conc. of acemannan
Codes sample (mg) (mg/10 mL)
AV 4 10 4.3 C7A (D) 10 4.6 C7A (L) 10 4.4 C7A*(D) 10 6.1 C7A*(L) 10 4.9
Note: C7A represents callus cultures in MS media supplemented with 2 mg/L NAA and 3% sucrose for. C7A* indicates in vitro plant regenerated in MS medium supplemented with a combination of 1.0 mg/L BAP and 0.1 mg/L IBA. Dark and light conditions are represented by (D) and (L) respectively.
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Analysis of acemannan content in conventionally propagated and in vitro regenerated Aloe vera plants
12 10 10 10 10
(mg) 8 6 4 6.1 2 4.3 4.9 sample 0 of
(L) gel (D)
vera
Weight (AG) AVRG AVRG Aloe Weight of sample mg Conc of Acemannan (mg/10 ml)
GRAPH 2: Shows the analysis of acemannan content in conventionally propagated and in vitro regenerated Aloe vera plants
3.3.4 Identity assessment of acemannan by ¹H‐NMR
In 1998 Diehl and Teichmuller documented that the identity of acemannan in Aloe vera gel preparations can be significantly assessed by ¹H-NMR. Acemannan is a linear polysaccharide composed by β-(1→4)-linked mannan partially acetylated in positions 2, 3 or 6. These acetyl groups generate a characteristics signal (2.0-2.3 ppm) in ¹H-NMR, which shows the characteristic presence of acemannan and is considered as the fingerprint of acemannan in Aloe vera (Diehl and Teichmuller,
1998;). Similarly, the results obtained by the ¹H-NMR spectra of Aloe vera standard
(Figure 9), plant gel (Figure 10) and callus (Figure 11) extracts confirm the presence of the immunomodulatory polysaccharide acemannan as shown below in Figure 8
(Bozzi et al., 2007; Krokida et al., 2011; Prerak et al., 2012; Ray et al., 2013).
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.
Figure 8: ¹H- NMR spectra of Aloe vera gel powder concentrate 200:1
(Bozzi, et al., 2007)
Figure 9: ¹H-NMR spectra of Aloe vera standard
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Figure 10: ¹H-NMR spectra of Aloe vera plant gel extract
Figure 11: ¹H-NMR spectra of Aloe vera callus extract
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3.3.5 Analysis of acemannan production and enhancement under different carbohydrate stress conditions
After optimization of several isolation and purification methods, protocols were optimized for qualitative and quantitative analysis of acemannan content in Aloe vera extracts. Acemannan production was confirmed in callus cultures by TLC analysis having Rf value of 0.6 which was similar to the standard compound. HPLC method was successfully employed and the quantitative analysis demonstrated the same retention time (2.0 min) when compared to the standard (Graph 3).
Analysis of Acemannan content under Carbohydrate Stress Conditions in Aloe vera Calli and Regenerated plants 12 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 9.5 (mg) 8 6.1 4.9 6 4.3 4.4 4.0 1.9 2.2 4 1.3 1.1 1.1 sample 0.7 1.0 2 0.4 0.1 0.6 0.2 0.5 0.5 0.1 0.1 0.3 0.3 0.1 of 0 (L) (C) gel… (D)
Weight Sucrose Sucrose Sucrose Sucrose Sucrose vera Glucose Glucose Glucose Glucose Glucose
AVRG AVRG Mannose Mannose Mannose Mannose Mannose Galactose Galactose Galactose Galactose Galactose
Control 2% 3% 4% 5% 6% 2% 3% 4% 5% 6% 2% 3% 4% 5% 6% Aloe 2% 3% 4% 5% 6%
Weight of sample mg "Conc. Of Acemannan (mg/10 ml)
Graph 3: Shows the combined analysis of acemannan content under carbohydrate stress conditions in Aloe vera calli and regenerated plant
HPLC analysis provides strong evidence for the production and quantification of bioactive compounds. The extracts of callus cultures subjected to different carbohydrate stress treatments and kept under dark conditions for 30 days were analyzed for acemannan content by HPLC and strong variations in the acemannan levels detected can be seen in Graph 3 and HPLC chromatograms. Of the different
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sugars and their concentrations tested, mannose and sucrose were found to be prominent in enhancing the production of this biologically potent polysaccharide.
The highest concentration of acemannan i.e 0.95 mg/mL was recorded in callus cultures incubated in media having 3% mannose sugar (Graph 5), which revealed a remarkable enhancement of acemannan production as compared to the in vivo propagation of Aloe vera plant in soil conditions having 0.43 mg/mL yield.
Calli cultured in media with 3% sucrose only increased minimal production of this compound in vitro giving 0.44 mg/mL yield (Graph 4). On the contrary, addition of glucose (Graph 6) and galactose (Graph 7) in the media proved to be least efficient and suppressed the yield of acemannan in all the tested concentrations.
Garro-Monge, et al., in 2008 reported acemannan detection by using high performance liquid chromatography (HPLC) in Aloe barbadensis Mill from inner gel and embryogenic calluses. According to their results the acemannan concentration in fresh leaves was much higher (85 mg/100 mL) than embryogenic calluses (0.8-2.1 mg/100 mL), when cultured on MS media having 3% sucrose and kept under dark.
Kim et al., in 2012 analyzed Aloe barbadensis suspension cultures for the production of extracellular polysaccharides and found a β-1,4- glucomannan, which is originally present in Aloe vera leaves. The polysaccharide existed in acetylated form having a molecular weight of 490 kD. Under optimal conditions maximum levels of callus growth (20.4 g/L) was achieved in which the production of extracellular polysaccharide was 2.5 g/L.
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Our experimental results showed a significant promoting effect of 3% mannose and
3% sucrose on callus cultured in dark conditions, stimulating acemannan concentration several folds (0.95 mg/mL and 0.44 mg/mL, respectively). Additionally, light also had a major effect on production of acemannan and the plants regenerated from callus in dark conditions cultured on MS media having 3% sucrose, showed higher levels (0.61 mg/mL) as compared to production in the in vitro plants under light conditions (0.49 mg/mL) and conventionally propagated Aloe vera plants in field (0.43 mg/mL). This effect was most probably due to the plant growth hormones and carbon source used in the culture media, suggesting utilization of tissue culture technology for higher production of acemannan.
Another notable point in this experiment was the effect of light; acemannan was produced in much quantity in plants regenerated under dark conditions. The reasons for these results need to be explored further and might include the high activity of the enzymes in the secondary metabolite pathway and carbon dioxide flux, suggesting the possibility as an alternative production method of Aloe vera bioactives.
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Analysis of acemannan content under sucrose stress treatment in Aloe vera calli
12 10 10 10 10 10 10 10
(mg) 8 6 4.4 4 1.3 1.1 0.7 2 0.4 0.1 sample 0 of
(C)
Sucrose Sucrose Sucrose Sucrose Sucrose
Weight
Control 2% 3% 4% 5% 6%
Weight of sample mg Conc of Acemannan (mg/10 ml)
Graph 4: Shows the analysis of acemannan content under sucrose stress conditions in Aloe vera calli.
Analysis of acemannan content under mannose stress treatment in Aloe vera calli
12 10 10 10 10 10 10 10
(mg) 8 9.5 6 1.9 4 1.1 0.6 2 0.4 4.0 sample 0 of
(C)
Mannose Mannose Mannose Mannose Mannose Weight
Control 2% 3% 4% 5% 6%
Weight of sample mg Conc of Acemannan (mg/10 ml)
Graph 5: Shows the analysis of acemannan content under mannose stress conditions in Aloe vera calli.
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Analysis of acemannan content under glucose stress treatment in Aloe vera calli
12 10 10 10 10 10 10 10
(mg) 8 6 2.2 4 1.0 2 0.4 0.2 0.5 0.5
sample 0 of
(C)
Glucose Glucose Glucose Glucose Glucose
Weight Control 2% 3% 4% 5% 6%
Weight of sample mg Conc of Acemannan (mg/10 ml)
Graph 6: Shows the analysis of acemannan content under glucose stress conditions in Aloe vera calli.
Analysis of acemannan content under galactose stress treatment in Aloe vera calli
15 10 10 10 10 10 10 10 (mg)
5 0.4 0.1 0.1 0.3 0.3 0.1 0 sample
of
(C) 2% 3% 4% 5% 6% Control Galactose Galactose Galactose Galactose Galactose Weight Weight of sample mg Conc of Acemannan (mg/10 ml)
Graph 7: Shows the analysis of acemannan content under galactose stress conditions in Aloe vera calli.
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3.3.6 Standard Calibration Plot
Quantification of Aloe vera acemannan was carried out at λ 209 nm. Linear calibration plot for acetyl-mannan was obtained at 10 different concentrations between 1-10 mg/10 mL. To check the reproducibility of results the data was obtained in triplicate.
Linear regression analysis of the calibration plot yielded equations Y = 670193x +
140549. The calibration data indicated (r²>0.9984) linearity of the detector response by HPLC-UV method. The results revealed excellent correlation between concentration and peak area (Graph 8). The linear calibration plot for acemannan obtained is shown below in Graph 8, which was then used to determine the concentration of acemannan in all the samples.
Calibration Curve y = 670193x + 140549 R² = 0.9984 8000000 6000000 4000000 Area 2000000 0 024681012 Amount
Graph 8: Shows the calibration curve of acemannan standard
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Graph 9: HPLC-UV chromatograms of Aloe vera standard, samples and control at λ 209 nm. The retention time of acemannan is at 2.1 min. The codes represented below indicate the Aloe vera varieties, in vitro grown Aloe barbadensis plants and callus cultures grown in MS medium supplemented with (2-6%) sucrose, mannose, glucose and galactose sugars.
1.60
1.40 2.333
1.20
1.00
AU 0.80
0.60
0.40
0.20
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.0 Minutes
HPLC-UV Chromatograms of Acemannan Standard
Control
0.12
0.10 2.062
0.08
0.06 AU
0.04
0.02 3.701 1.501 0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.0 Minutes
AC3S (L): Aloe barbadensis callus cultures under light conditions on 3% sucrose in culture media
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1.80
1.60 2.156 1.40
1.20
1.00
AU 0.80
0.60
0.40 1.775 2.786 0.20 3.777
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.0 Minutes
AV (1): Aloe vera variety 1 AV (2): Aloe vera variety 2
0.090 1.40
0.080 2.145
2.088 1.20 0.070 1.00 0.060 2.870 0.050 0.80 AU AU 0.040 0.60 3.627 0.030 0.40
0.020 1.551 0.20 0.010 2.766 1.516 0.000 0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.0 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.0 Minutes Minutes
AV (3): Aloe vera variety 3 AV (4): Aloe vera variety 4
0.16 0.60 0.14 2.328 2.042 0.50 0.12
0.10 0.40
0.08 AU AU 0.30
0.06 0.20
0.04 2.773 3.207 1.892 0.10 0.02
0.00 0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.0 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.0 Minutes Minutes
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AVRG (D): In vitro regenerated Aloe vera plants under dark.
2.00
1.80 2.204 1.60
1.40
1.20
1.00 AU
0.80 2.709
0.60 3.749
0.40
0.20
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.5 Minutes
AVRG (L): In vitro regenerated Aloe vera plants under light.
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Aloe vera callus cultures grown in MS medium supplemented with (2-6%) Galactose
2% Galactose 3% Galactose
0.025 0.025 2.066 2.031 0.020 0.020
0.015 0.015 AU AU
0.010 0.010 2.832 2.642 3.157 1.524 1.503 3.141 0.005 0.005
0.000 0.000
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5. 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5. Minutes Minutes
4% Galactose 5% Galactose
0.12
0.12 2.105 0.10 2.140 0.10
0.08 0.08
0.06 AU AU 0.06
0.04 0.04
0.02 0.02 1.511 3.154 2.855 3.152 1.531 1.262 0.00 0.00 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5. 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.0 Minutes Minutes
6% Galactose
0.025 2.074
0.020
0.015 AU
0.010 3.112 3.821 2.897
0.005 1.503 0.860
0.000
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.0 Minutes
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Aloe vera callus cultures grown in MS medium supplemented with (2-6%) Mannose
2% mannose 3% Mannose
1.80 2.00
1.60 1.80 2.108 2.122
1.40 1.60 1.40 1.20 1.20 1.00
AU 1.00 AU 0.80 0.80
0.60 0.60 2.416 2.696 0.40 0.40 3.416 3.317 4.675
0.20 4.732 0.20 1.666 0.00 0.00 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.5 Minutes Minutes
4% Mannose 5% Mannose
0.70 0.50 2.105 0.60 2.083 0.40
0.50 0.30 0.40 AU AU 0.30 0.20
0.20 0.10 2.813 0.10 2.607 3.548 3.542 4.869 3.955 4.854 0.00 0.00 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.0 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.0 Minutes Minutes
6% Mannose
0.16
0.14 2.107 0.12
0.10
0.08 2.885 AU
0.06
0.04 3.502 0.02 4.857
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.0 Minutes
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Aloe vera callus cultures grown in MS medium supplemented with (2-6%) Sucrose
2% Sucrose 3% Sucrose
1.40 2.145 0.40 2.092 1.20
1.00 0.30
0.80 AU AU 0.20 0.60
0.40 0.10 0.20 2.565 2.766 1.471 3.691 1.516 0.00 0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.0 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.0 Minutes Minutes
4% Sucrose 5% Sucrose
0.035
0.30 0.030 2.075 2.095
0.25 0.025
0.20 0.020 2.653 AU AU 0.015 0.15
0.010 0.10
0.005 0.05 1.488 4.423 1.158 0.854 0.000 0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5. 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.0 Minutes Minutes
6% Sucrose
0.050 2.065 2.762
0.040 2.507 3.415 0.030 AU
0.020
0.010 1.508 1.185
0.000
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.0 Minutes
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Aloe vera callus cultures grown in MS medium supplemented with (2-6%) Glucose
2% Glucose 3% Glucose
0.20 0.10 0.18 2.054 2.024 0.08 0.16 0.14
0.06 0.12 0.10 AU AU
0.04 0.08
0.06
0.02 0.04 2.726 2.710 1.430 0.02 3.379 3.780 1.475 1.203 0.00 0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.0 Minutes Minutes
4% Glucose 5% Glucose
0.90 0.40
0.80
0.35 2.076 2.102 0.70 0.30 0.60 0.25 0.50
AU 0.20 AU 0.40 0.15 0.30 0.10 0.20
0.05 2.741 0.10 2.617 3.523 1.531 1.593 3.645 4.080 4.922 0.00 0.00
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CHAPTER 4
BIOTRANSFORMATION
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4.1 INTRODUCTION
4.1.1 Biotransformation
The chemical modifications in the medicinally important compounds carried out by living cells, organs or enzymes are known as biotransformation, which has a great potential to generate novel products of high medicinal importance. These compounds can be chemically synthesized but the process might be of high cost and labor intensive (Schulze & Wabbolts, 1999). Biotransformation can be done by using variety of living cells including microbial (Ward & Singh, 2000), fungal (Ramachandra &
Ravishankar, 2000), plant (Saifullah et al., 2011; Choudhary et al., 2006), animal
(Novais, 1998), or even isolated enzymes (Franssen & Walton, 1999) can also be used. The use of microbial and fungal cell for the biotransformation has been extensively applied but the enzyme system of these sources is limited for certain types of chemical modifications (Ward & Singh, 2000) while the animal cells or the isolated enzymes are highly sensitive and difficult to handle (Franssen & Walton, 1999).
Adrenosterone (1) is a hormone that is secreted by the adrenal gland in the human body. It is also produced in the testes of some fish like Salmon and Talpia, along with another major hormone; androgen 11-ketotestosterone. Its function in human is to modulate the presence of Cortisol (a vital hormone for efficient fuel processing under stress conditions) in certain body fluids.
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4.2 MATERIALS AND METHODS
4.2.1 Establishment of Plant Cell Culture
The media used for the induction of calli were found insufficient for the establishment of suspension cultures due to the lack of disintegration of calli clumps. To resolve this problem, MS media with higher concentrations of NAA (2.0, 4.0, 6.0, 8.0 10.0, 12.0 and 14.0 mg/L) along with varied concentrations of BAP (0.5, 1.0 and 1.5 mg/L) were examined. Each media formulation was inoculated by 5 gm of calli. Erlenmyer flasks of 250 mL volume, each containing 50 mL of MS liquid media were used in this experiment. The cultures were kept at 22 ± 2º C in light conditions. The flasks were kept on an orbital shaker and rotate at 100 rpm. The transferred clumps of calli were visually monitored for 15 days and on the basis of their disintegration, best media formulation was selected.
4.2.2 Growth Curve Analysis
Medium selected for the cell suspension cultures was used for the growth curve analysis. Freshly grown calli (5 gm) were transferred to 24 Erlenmyer flasks (250 mL) containing 50 mL of medium (MS media with 12.0 mg/L NAA and 1.0 mg/L BAP). All the flasks were kept at 22 ± 2 ºC in light conditions same as mentioned above with an agitation on an orbital shaker (100 rpm). After every five days, three flasks were harvested and their mean Fresh Weight (FW) was calculated.
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4.2.3 Biotransformation of Adrenosterone
During the biotransformation experiment, 800 ml of a 15 days old suspension was inoculated with 200 mg of substrate (Compound 1). The suspension was kept on shaker with continuous shaking at 100 rpm incubated at 22±2ºC with 16/8 hrs photoperiod. After every five days, the degree of conversion was measured using
TLC. After twenty days, as the TLC showed no spot of Compound 1, the cells were separated from the nutrient medium by filtration. The cells were washed with CH2Cl2
(200 ml) and the filtrate was extracted with CH2Cl2 (3 200 ml). The combined organic extract was dried over anhydrous Na2SO4, evaporated under reduced pressure, which afforded a brown gum (0.41 g), which after repeated CC (petroleum ether/ AcOEt gradient) yielded compounds: 1 (51.4 mg; with petroleum ether/AcOEt
72: 28), 2 (16.2 mg; with petroleum ether/AcOEt 61: 39), and 3 (81.7 mg; with petroleum ether/AcOEt 57: 43).
Two Controls were also run along with the test. One is to check the substrate’s stability (only the media for suspension culture was fed with the substrate, without any calli) and the other to identify the metabolites produced by the plant cell itself (Cell suspension culture without any substrate feeding). TLC indexing was performed to nullify the spots appeared due to substrate degradation or plant metabolites, if any.
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4.3 RESULTS AND DISCUSSION
4.3.1 Suspension Cultures and Growth Curve Analysis
In search of the medium suitable for the maximum disintegration of the calli clumps,
MS media with 12.0 mg/L of NAA and 1.0 mg/L of BAP was found best. All the cultures were maintained at the temperature of 22 ± 20C, under white light with 16/8 hrs photoperiod. After the inoculation of calli, as all the media formulations were visually monitored, this formulation showed maximum disintegration of the clumps and a very efficient suspension culture having an evenly distributed plant cells was formed after just five or six days of inoculation.
The same medium was used for the growth curve analysis and the cultures were kept under the same conditions as mentioned above. The mean FW was calculated after every fifth day and a graph was plotted, showing the change in FW of the suspension with respect to time. As Graph-9 shows the lag phase of the growth curve which lasted for atleast five days. The cells were grown exponentially from seventh to twenty fifth day of inoculation and after that the growth of the suspension cultures entered into the stationary phase. As the cells are in maximum of their functionality and growth during the log phase, the starting of the log phase (fifth day) was selected for the inoculation of substrate to the suspension culture for biotransformation.
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30.00
25.00
20.00
15.00
10.00 Fresh Wieght (gm) Wieght Fresh 5.00 0.00 1 5 10 15 20 25 30 35 Days
Graph 10: Growth curve analysis in the suspension culture of Aloe barbadensis.
4.3.2 Biotransformation of adrenosterone
Cell suspension culture of Aloe barbadensis, maintained at the same temperature, light and agitation conditions as stated above, showed three types of reactions when compound 1 was incubated with cell suspension culture of A.barbadensis included the formation of C=C moiety at C-1/C-2 and yielded 1-2-dehydroadrenosterone (2), reduction of C-4/C-5 olefinic double bond resulted the formation of 5-androst-1-ene-
3,11,17-trione (3) and the reduction of C-17 keto group, which gave 17- hydroxyandrost-4-ene-3,11-dione (4) (Scheme-1). Structures of transformed metabolites were elucidated through comparison of their reported data (Musharaf et al., 2002). Compounds 2 and 4 were previously reported as metabolites of 1, with various fungal biotransformations (Musharaf et al., 2002; Greca et al., 1997).
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O 18 O 12 17 16 19 11 13 H 14 15
1 9 10 8 2 H H 3 5 4 6 7 O 1
O O OH O O O
H H H H H H
O O O H 4 2 3
Scheme 1: Biotransformation of compound 1 by Cell suspension culture of Aloe barbadensis
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CHAPTER 5
MOLECULAR ANALYSIS
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5.1 INTRODUCTION
5.1.1 Molecular Analysis of Aloe vera plants by Genetic/Molecular
Markers
Plant tissue culture techniques are known to induce somaclonal variations. Frequency of these variations differs with the source of explants, their regeneration methods, composition of culture medium and cultural conditions. The first observation of somaclonal variations was reported by Braun in 1959. Advances in molecular biology have revolutionized every field of biological sciences such as, DNA based markers have been used for individual identification, genome mapping, pedigree and phylogenetic diversity analysis in numerous taxa and in selecting somaclonal variations. Molecular biological tools can accelerate artificial breeding processes
(Smolik et al., 2009) and clarify the genetic mechanisms that cannot be easily detected with plant breeding techniques (Staub et al., 1996 and Gupta et al., 2010).
Molecular tools can also give important information about the genetic distances between species (Mihalte et al., 2011).
A genetic marker is “a gene or DNA sequence with a known location on a chromosome and associated with a particular gene or a character”. It can be described as a variation, which may arise due to mutation or alteration of nucleotide in the genomic loci that can be observed. DNA-based molecular marker helps in improvement of plant species. DNA markers are more reliable because the genetic information is unique for each species and is independent of age, physiological conditions and environmental factors. The molecular marker technique efficiency is based on the amount of polymorphism, it can detect in the given accessions
(Tharachand et al., 2012). Molecular marker studies on Aloe vera have been
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published by using the following molecular techniques such as: AFLP (Amplified
Fragment Length Polymorphism) (Tripathi et al., 2011), RAPD (Random Amplified
Polymorphic DNA), (Nayanakantha et al., 2010) and ISSR (Inter Simple Sequence
Repeats), Rathore et al., (2011).
The increasing development and generalized use of a large number of methodologies during the last years, requires comparative studies in order to choose the best DNA marker technology to be used in fingerprinting and in diversity studies, considering reproducibility, costs, sensibility and level of polymorphisms detection. Molecular technique comparisons have become important because, depending on the objective of the study, one technique can be more appropriate than another, as well as different techniques being informative at different taxonomic levels. However, the similarity among the different molecular techniques began to be discussed with opposite results among authors. Several works report comparable results among different markers while others show considerable variations (Goulao & Oliveira, 2001).
There is a range of molecular marker types available, but the choice of an appropriate genetic marker depends on the experience and competence of the researchers, also on laboratory facilities (Benali et al., 2011). Since 1950s a variety of molecular markers has become available and has been applied to many areas in biology such as population genetics, forensics, paternity testing and gene mapping. However, it is only since the 1980s that these molecular markers have become widely used in the field of conservation biology. In conclusion, the detection and quantification of genetic diversity/similarity in plant species is important for successful conservation of genetic resources and plant breeding (Geleta et al., 2012). Plant genetic diversity is highly essential for genetic improvement of crops for sustainable agriculture and its gradual
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loss is as a consequent to rapid human population growth, industrialization, deforestation and natural calamities.
5.1.2 Random Amplified Polymorphic DNA (RAPD)
Molecular markers have overcome limitations of morphological and biochemical markers due to the influence of environment on the performance of genotypes. A wide range of molecular markers have been used to assess genetic diversity of Aloe vera.
The polymerase Chain Reaction (PCR) is an in vitro enzymatic method of amplifying specific DNA sequences which was conceived originally by Kary Mullis in 1983 (Mullis
& Faloona, 1987). The PCR technology hinges on the availability of DNA polymerase
(Taq polymerase) from the Thermophilic bacterium Thermus aquaticus which retains activity even after prolonged incubation at 1000C which denature double stranded
DNA templates. Even minute quantities of target DNA can be specifically amplified by supplying Taq polymerase, excess nucleotides and oligonucleotide primers are used to anneal with complementary sequence and finally activate the DNA polymerase
(Aitchitt, 1995). Random Amplified Polymorphic DNA (RAPD) is powerful technique considered as a useful tool, it need tiny amounts of DNA to give rapid and accurate identification of alien species especially in the developing countries, where DNA based methods are unavailable due to their high cost, the requirement for complex equipments and expertise (El-Mergawy et al., 2011) Among molecular markers
RAPDs are the most widely applied most probably because they do not require the knowledge of genomic sequences and also the protocol is relatively simple, rapid and cost effective (Bornet et al., 2004).
Random Amplified Polymorphic DNA (RAPD) technique can be used to identify and determine plant genomes or to estimate the phylogenetic relationship among the
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individual genomes of Aloe vera plants (Nayanakantha et al., 2010) and it is considered a fast and easy approach to solve the obscured identification problems.
Many kinds of plants have been fingerprinted using RAPD markers to evaluate their phylogenetic relationship, genetic variability and genetic relationships (Sangin et. al.,
2006). Random amplified polymorphic DNA (RAPD) method requires no prior knowledge of DNA sequence. The DNA fragments are amplified by the polymerase chain reaction (PCR) using short (usually 10bp) synthetic primers of a random sequence. Polymorphism among the amplification products are useful genetic markers and can be detected through examination of Ethidium Bromide-stained agarose gel. RAPD on the other hand, require only small amounts of DNA sample without involving radioactive labels and is simple as well as fast technique. RAPD has proven to be quite efficient for assessment/detecting genetic fidelity (Harirah and
Khalid, 2006) or variations, even in closely related organisms such as two near isogenic lines (NIL) (Kumar et al., 2010). Genetic diversity is the basis of plant breeding, so understanding and assessing it is important for crop management, crop improvement by selection, use of crop germplasm, detection of genome structure, and transfer of desirable traits to other plants (Sohrabi et al., 2012, Williams et al., 1990,
Welsh & McClelland, 1990 and INBAP, 1996).
5.1.3 Inter Simple Sequence Repeats (ISSR)
The Inter Simple Sequence Repeats (ISSR) was first developed by Zietkiewicz et al.,
(1994) to rapidly differentiate between closely related individuals. This technique is known with other names such as, MP-PCR (microsatellite-primed PCR) or ISA (Inter-
SSR Amplification) or RAMP (Randomly Amplified Microsatellite Amplification) marker system. ISSR technique is also a very simple, fast, cost-effective, highly discriminative and reliable (Kumar et al., 2010). The ISSR is a PCR based molecular biology
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technique, which involves amplification of DNA segments present at an amplifiable distance in between two identical microsatellite repeat regions oriented in opposite direction. Inter-SSRs are semi-arbitrary markers amplified by PCR in the presence of one or two primers complementary to a target microsatellite. Similar to RAPD markers, ISSR markers do not require genome sequence information but provide multi-loci amplification (Tran, 2005).
ISSR markers were developed from the need to explore microsatellite repeats without the use of DNA sequencing. The technique is based on the amplification of DNA segments between two microsatellite repeated regions. ISSR is a simple, fast and an efficient technique that produces amplified products of 200–2000 bp in length. The technique is highly reproducible due to the use of longer primers, which allow for high annealing temperatures. The technique uses a primer containing only the repetition of a particular SSR (di-, tri-, tetra-, or pentanucleotides), of 16–25 bp, either unanchored or anchored in the 3' or 5' ends for up to four nucleotides, which are often degenerate.
The ISSR combines the advantages of AFLP markers and SSR with the convenience of RAPD. This technique has been used successfully for diversity analysis in several fruit species, such as trifoliate orange, citrus, diploid banana, poolasan hairy litchi and grape (Santos et al., 2011).
ISSR is modification of SSR-based marker systems; inter simple sequence repeat
(ISSR); is a new technique (Tahery, 2012) and have been successfully applied in many crop species (Belaid et al., 2006). It requires very small amount of template
DNA and is convenient in result recording and highly reproducible. This technique is widely used in species identification, genetic mapping, gene locating, phylogeny and evolution (Lijun & Xuexiao, 2012).
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ISSR markers are known to be more reproducible than RAPD markers and they have been successfully applied to the study of genetic diversity in plants (Bornet et al.,
2002). First, it permits detection of polymorphisms in microsatellites and intermicrosatellites loci without previous knowledge of the DNA sequence.
Microsatellite regions are abundant throughout the eukaryotic genome, which are highly polymorphic in length and are interspersed. Secondly, ISSR is informative about many loci and are suitable to discriminate closely related genotype variants.
And lastly, ISSR markers constitute discrete markers suitable in the DNA fingerprinting (Zehdi et al., 2004). Inter Simple sequence repeats (ISSR) marker assays were employed to assess the genetic stability of tissue cultured Aloe vera plantlets, to validate the most reliable regeneration method for true-to-type propagation of sweet aloe (Rathore et al., 2011).
ISSR is considered to achieve higher reproducibility than RAPD markers and is repeated to detect a high portion of genomic variation than RFLP (Konate et al.,
2009). They are easy to handle, highly informative and repeatable. Since repeated sequences are abundant throughout the genome, SSR primers anneal in several regions typically giving a complex amplification pattern in which fragments are often polymorphic between different individuals. Thus, ISSR DNA has been proposed as a new source of genetic marker that overcomes many of the technical limitations of
RFLP and RAPD analyses, even in plants (Nagaoka & Ogihara, 1997).
The technique has already been used in plants and is useful to study genetic diversity, phylogenetic studies, gene tagging, genome mapping in wide range of crop species and to distinguish among cultivars and to construct linkage maps (Moreno, et al., 1998 and Reddy et al., 2002). In higher plants or animals, Inter Simple Sequence Repeat or
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ISSR markers are more and more in demand, because they are known to be abundant, very reproducible, highly polymorphic, highly informative and quick to use
(Bornet et al., 2004).
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5.2 MATERIALS AND METHODS
5.2.1 Evaluation of Genetic stability of Aloe vera plants by RAPD and
ISSR Marker Assays
Molecular studies using RAPD and ISSR were carried out for the assessment of genetic stability and variability of in vivo mother plant and in vitro regenerated plantlets of Aloe vera L.
5.2.2 Plant Material
The leaves of conventionally grown Aloe vera plants were collected from nursery of
H.E.J Research Institute of Chemistry and leaves of in vitro propagated plants were taken from the growth room of Plant Tissue Culture and Biotechnology Wing.
5.2.3 DNA extraction
The Aloe vera plant DNA was extracted using (CTAB) method. Plant material was grinded using liquid nitrogen. Liquid nitrogen is frequently used in DNA extraction protocols as it facilitates grinding of the tissue by turning it into solid form and has an additional advantage of maintaining low temperature. Many small laboratories of developing countries faced the problem of unavailability of liquid nitrogen. Storage and maintenance of liquid nitrogen is also difficult. The highly versatile Cetyl trimethyl ammonium bromide (CTAB) method (Sambrok, et al., 1989) is a standard method for the extraction of DNA from various plant materials. The availability of high-quality genomic DNA is a crucial prerequisite for molecular genetic analysis of crops. There are three main contaminants associated with plant DNA that can cause considerable difficulties when conducting PCR experiments: polyphenolic compounds, polysaccharides and RNA. Extraction of intact, high molecular-weight DNA that can
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support PCR, genomic blot analysis, fingerprinting and other molecular analysis is a challenge when the plant tissue is rich in polysaccharides, secondary metabolites or polyphenolics.
It is necessary to isolate good quality DNA that is relatively free from many contaminants found in plant cells. Many plant species contain characteristically high amounts of proteins, polysaccharides, polyphenols (Angeles et al., 2005) and other secondary metabolites, substance known for binding firmly to nucleic acids during
DNA extraction and interfering with subsequent reactions (Ribeiro and Lovato, 2007).
Total cellular/genomic DNA isolation was performed by classical cetyltrimethyl ammonium bromide (CTAB) method described by Doyle and Doyle (1990) with some modifications. To obtain good quality DNA, the utilization of fresh and young leaf tissue is ideal (Ribeiro and Lovato, 2007). Latex gloves were worn constantly during the DNA extraction to minimize the risk of contamination of samples with nucleases from the skin and to protect the skin from hazardous chemicals such as phenol, chloroform and liquid nitrogen. Goggles were worn during the procedure to protect eyes especially during grinding. About 1.5 g of Aloe vera leaf samples were cut into small pieces and ground to fine powder in pre-chilled pestle and mortar. Fine powder was suspended/homogenized into 2 mL preheated (65°C) CTAB buffer (2% CTAB,
1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl, pH 8.0 and 2% mercaptoethanol), kept on hot plate at 600C for 40 minutes to homogenize and poured into 15 mL falcon tubes. The homogenate was incubated at 65°C for 1 hour in shaking water bath after then 2 ml of fresh chloroform: iso-amyl alcohol (24:1, v/v) was added and gently mixed by inverting the tubes for 50 times until there was no interface. After then mixture was centrifuged (Eppendorf® Centrifuge 5804R) at 10,000 rpm for 10 min at 40C to separate the phases. The supernatant/aqueous phase was collected in other falcon
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tubes and 1:1 ratio of ice cold isopropanol was added to precipitate the DNA.
Isopropanol was mixed by shaking the tube, when precipitation was not obvious the samples were kept at -200C for one an hour in freezer. After 1 hour the DNA was spooled with the help of wire loop. In case, if DNA was not spool able then it was again centrifuged at 10,000 rpm for 2-3 min at 40C. The DNA made pellet at the bottom of the tube. The spooled DNA was put into 5 mL washing buffer for 20 min,
DNA was collected from washing buffer, air dried and then re-suspended in TE buffer into labeled microfuge tubes. The DNA dissolved/eluted into 1 mL TE buffer, vortexed and left for overnight. Upon extraction of total genomic DNA of each cultivar was given an extraction code and transferred to eppendroff tubes.
Next day DNA samples were vortexed, added 10 µl RNase A and incubated at 370C for 20 min. After incubation the samples were centrifuged (Biofuge pico Heraeus) at
13,000 rpm for 10 min. The supernatant was extracted; finally DNA quantification was detected on 0.8% agarose gel (Wang et al., 2012) and by UV spectrophotometer. All the coded eppendorff tubes were kept in the freezer at – 20, at Molecular Biology and
Quality Control Laboratory, Plant Tissue Culture and Biotechnology Wing,
International Center for Chemical and Biological Sciences, University of Karachi-
Karachi-75270, Pakistan. The remaining leaf samples were kept in -200C freezer.
5.2.4 DNA quantification
The nucleic acid concentration was determined using a spectrophotometer
(Schimadzu, 2000). Silica (Quartz) Ultra Micro Spectrophotometer cuvette was used for holding the samples and T.E buffer (10 mM Tris Hcl (pH 7.5), 1 mM EDTA) solution was used as a standard/blank for calibrations of the spectrophotometer at 260 nm and 280 nm. The DNA concentrations were measured in spectrophotometer using
1:1000 dilutions. Readings were recorded for each DNA sample at wavelength of 260
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nm and 280 nm. An O.D. of 1 corresponded to approximately 50µg ml-1 double stranded DNA. The ratio of the readings was noted at 260 nm and 280 nm provided an estimation of the purity of nucleic acids. The readings at 260 nm were used to calculate the DNA concentration in the samples, by using the formula:
DNA concentration (µg/ml) = A260 x 50 x dilution factor (100)
An optical density value 1.0 corresponds to approximately 50 µg/mL for double stranded DNA. Pure samples of DNA have ratios of 1.8 to 2.0 at O.D. 260/O.D. 280 and values of less than this for DNA samples of lower purity. If there is contamination of protein or phenols the ratio will be significantly lower than 1.8 and higher ratio signify contamination by RNA (Sambrook et al., 1989). Besides this conventional method of DNA estimation was performed by gel on 0.8% with λ DNA. The amount of plant genomic DNA was estimated by visual comparison of band intensity between λ
DNA and plant DNA under following ethidium bromide staining. DNA samples were diluted in T.E buffer to a working concentration of approximately 10 ng µl-1.
5.2.5 Primers
The sequences of the different primers RAPD (octamer oligonucleotide) used in this study are given in Tables 10b.
5.2.6 Molecular Marker
Molecular size of PCR amplified products were estimated by using 1kb (Invitrogen) and 100 bp (Fermentas) DNA ladder.
5.2.7 DNA Amplification by RAPD‐PCR
PCR amplification was carried out in Hi-Temp 96 wells Thermal cycler (Master Cycler,
Eppendorf, Germany). The following concentration of PCR reagents (Fermentas,
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USA) were used for 25 µl final reaction volume (Table 10a). The cycling parameters consisted of 30 min denaturation at 940C, followed by 45 cycles of 30 sec at 940C, 1 min at 320C, and 2 min at 720C (denaturation, annealing and extension).
Table 10a: Composition of master mix for RAPD-PCR reaction
Reagents Concentration Volume PCR Mastermix 2x 12.5uL MgCl 25mM 1.5 µL Primer 4 µM 2.0 µL DNA template 10ng/ µl 2.5 µL
ddH2O ----- 6.5uL Total volume 25.0 µl
The reaction was finally incubated at 720C for 4 min, followed by soaking period at 40C until recovery. The lid temperature was kept constant at 1090C.
Table 10b: Primer sequence used for the RAPD-PCR reactions
No. Primer Name Primer Base Sequence 1. LC-76 GTGACGTAGG 2. LC-77 GGGTAACGCC 3. LC-81 AGCCAGCGAA 4. LC-83 AGCCAGCGAA 5. LC-87 AGGTGACCGT 6. LC-90 GTGAGGCGTC 7. OPL-1 GGCATGACCT 6. OPQ-12 AGTAGGGCAC
5.2.8 Agarose Gel Electrophoresis Gel agarose was prepared in order to verify the presence and the size of PCR products (amplicons) and 1 Kb and 100 bp markers were used to calculate the
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molecular weight of the amplified PCR products. The procedure was performed as following:
5.2.8.1 Melting of the Agar Molecular Biology grade agarose (Gene-Link and Scharlu) was used at 1 % for the separation of amplified PCR products. The agarose was suspended in the 1x TBE
(Tris Borate EDTA) buffer pH 8.0 in a flask. The flask was covered with another small mouth flask as the vapors can come back again in the same flask of agarose. The solution was heated in microwave oven (Sharp 1000W/R-2197), till the agarose dissolved completely and solution become clear. During boiling the solution was gently swirled to re-suspend any settled agarose. The flask was removed carefully using thick hand gloves/protector. Then gels were allowed to cool at about 600C, as the flask can be handled comfortably. 5 µl ethidium bromide (0.05 µg/mL) was mixed properly in the agarose solution.
5.2.8.2 Pouring the Gel The electrophoresis gel tray/plate was prepared and a comb was placed in it to make loading wells. The cooled agarose gel was poured/casted into gel tray containing a comb. The agarose should come at least half way up the comb teeth. The agarose gels were allowed to solidify for 40 min after than comb was removed.
5.2.8.3 Setting up the Gel Tank After solidification gel was placed in the appropriate electrophoresis tank, the wells were placed to negative electrode and covered with 1X TBE running buffer. The DNA samples/PCR products were mixed with gel loading dye (0.25% (w/v) Bromophenol
Blue, 40% (w/v) sucrose, 0.1mM tris, and 0.05 mM EDTA) in a 1:4 ratio. The bromophenol blue dye give density to PCR product as the sample can sink to the bottom of the well properly and PCR product 8 µl plus 2 µl of loading dye mixed
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properly and loaded into each well created by comb. One (1) Kb ladder (Life
TechnologiesTM) used as molecular size marker or gene ruler on each gel.
5.2.8.4 Running and Analyzing the Gel The lid was placed on the tank and the electrode leads were connected to the power supply The electrophoresis was generally conducted by using large submarine units
(Thermo EC-320) at 60V for 2hr (Thermo EC, EC-250-90). Then gels were removed from the electrophoresis tank and placed in gel documentation system (UV TechTM,
UK), images of DNA bands obtained and photographed. The photos were saved in specific file till to be used. The PCR reactions were repeated twice for the confirmation of produced results.
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5.3 RESULTS AND DISCUSSION
5.3.1 Evaluation of Genetic Stability of Aloe vera plants by RAPD
Marker Assay
Among multilocus markers RAPD and ISSR techniques have been widely used to detect, evaluate and identify changes in the DNA sequence caused by somaclonal variation (Martins et al., 2004; Bhatia et al., 2009). Molecular characterization using
PCR based technique Random Amplified polymorphic DNA (RAPD) was conducted on conventionally grown (wild type) and tissue cultured Aloe vera plants to compare their genetic similarity/diversity. Eight primers were selected for the analysis as they were reported to produce polymorphic amplicons as shown in Figure 10 and Table
11a .
The primer LC-76 generated total 2 monomorphic bands with DNA templates of both conventional and tissue cultured plants and showed 0% polymorphism. Both Primers
LC-77 and LC-83 produced total 4 bands with 3 monomorphic and 1 polymorphic band, each exhibiting 25% polymorphism. Primer LC-81 produced all 4 monomorphic bands and no polymorphism was seen. LC-87 produced total 5 band with 3 monomorphic and 2 polymorphic amplicons, showing 40% polymorphism. While LC-
90 produced total 5 amplicons with 4 monomorphic and 1 polymorphic bands giving
20% polymorphism. OPL-1 generated 5 monomorphic bands with no polymorphism.
OPQ-12 produced highest number of bands (9), of which 6 were monomorphic and 3 polymorphic exhibiting 66.6% polymorphism. Thus with both types of Aloe vera DNA templates, all the eight RAPD primers generated 4.75 amplicons on the average, of which 3.75 were monomorphic and 1 polymorphic, showing 22% polymorphism.
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The DNA bands produced by individual wild type and in vitro cultured plants with all the eight bands was also calculated (Table 11a). With all the eight primers tested, conventionally grown Aloe vera plants produced total 29 band, showing minimum 1 and maximum 6 bands. However DNA templates of tissue cultured plants generated
32 bands with all the primers giving minimum 1 and maximum 9 bands.
Rathore et al., in 2011, published the first report on the comparison of genetic stability/instability of tissue-culture Aloe vera plants exploring two regeneration systems. No polymorphism was observed in regenerates produced from direct regeneration system from axillary buds, whereas 80% polymorphism was observed in plants produced through indirect regeneration via intermediate callus phase. Our results are in harmony with the previous findings, as in our Aloe vera plant regenerants produced through indirect regeneration via callus phase, have exhibited polymorphism in a range of 20-33% (Table 11b).
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Figure 12: RAPD profiles of Aloe vera wild type soil grown and tissue cultured plants with primers LC-76, LC-77, LC-81, LC-83, LC-87, LC-90, OPL-1 and OPQ-12.
LC-76 LC-77
LC-83 LC-81
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LC-90 LC-81
OPQ-12 OPL-1
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Table 11a: Total number of bands calculated from tissue cultured and wild type Aloe vera plant with eight RAPD primers
Primers Tissue Culture Wild type Total Bands
LC‐76 01 01 02 LC‐77 03 04 07 LC‐81 02 02 04 LC‐83 04 03 07 LC‐87 03 04 07 LC‐90 05 04 09 OPL‐1 05 05 10 OPQ‐12 09 06 15 Total 32 29 59
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Table 11b: Polymorphism percentage between tissue cultured and soil grown Aloe vera plant by using 8 RAPD primers
Sequence of primer Annealing MW (μg/μmole) GC Total bands Monomorphic Polymorphic Polymorphism % 5'‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐3' temperature Contents % bands bands LC‐76‐ GTGACGTAGG 410C 3,108.09 60 02 02 0 0
LC‐77‐ GGGTAACGCC 410C 3,053.05 70 04 03 01 25
LC‐81‐ AGCCAGCGAA 410C 3,046.07 60 04 04 0 0
LC‐83‐ AGCCAGCGAA 410C 3,046.07 60 04 03 01 25
LC‐87‐ AGGTGACCGT 410C 3,068.06 60 05 03 02 40
LC‐90‐ GTGAGGCGTC 410C 3,084.06 70 05 04 01 20
OPL‐1‐GGCATGACCT 320C 3,028.03 60 05 05 0 0
OPQ‐12‐AGTAGGGCAC 320C 3,077.08 60 09 06 03 33.3
Total 38 30 8 ‐
Average 4.75 3.75 1 18
Range 02‐09 02‐06 01‐03 20‐33.3
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5.3.2 Evaluation of Genetic Stability of Aloe vera plants by ISSR
Marker Assay
During the last decade new development in the PCR based techniques on DNA markers are used as powerful tools to validate the genetic fidelity of in vitro plants.
The present study was aimed at analyzing the genetic similarity and diversity in wild type field gown and tissue cultured Aloe vera plants by using ISSR markers. ISSR is a type of molecular markers based on inter-tandem repeats of short DNA sequences.
These inter repeats are highly polymorphic, even among closely related genotypes, due to the lack of functional constraints in these nonfunctioning regions. ISSR markers are simple, more reliable and proved highly efficient in analysis of genetic diversity studies (Nookaraju and Agrawal, 2012). The analysis was carried out with 4 inter simple sequence repeat (ISSR) markers, which were screened out for their effectiveness and reproducibility. The primers successfully produced clear bands with all the DNA templates of Aloe vera (Table 12a and Figure 11) as shown below.
Figure 13: ISSR profiles of Aloe vera wild type soil grown and tissue cultured plants with primers ISSR-A, ISSR-G, ISSR-M and ISSR-O.
142 Chapter 5 Molecular Analysis
Table 12a: Total number of bands calculated from Tissue culture and Wild type Aloe vera plant with four ISSR primers
Primers Tissue Culture Wild Total Bands ISSR‐A 06 06 12 ISSR‐G 04 03 07 ISSR‐M 04 04 08 ISSR‐O 04 04 08 Total 18 17 35
Tissue cultured plants produced total 18 bands with all the primers and 17 bands of wild type plants were produced. Of all the primers tested only primer ISSR-G exhibited
25% polymorphism with 3 monomorphic and 1 polymorphic band. The other primers did not produce any polymorphic band. On the average 4.25 monomorphic band were generated with all the four markers with a range of minimum 3 and maximum 6 amplicons (Table 12b).
143 Chapter 5 Molecular Analysis
Table 12b: Polymorphism percentage between Tissue culture and Wild type Aloe vera plant by using 4 ISSR primers
Sequence of primer Annealing Total bands Monomorphic Polymorphic Polymorphism temperature bands % 5'‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐3' bands
ISSR‐A‐ (CA)6GG 37.40C 06 06 0 0
ISSR‐G ‐(GT)6CC 37.40C 04 03 01 25
ISSR‐M‐ (AC)8T 37.40C 04 04 0 0
ISSR‐O ‐(AG)8T 37.40C 04 04 0 0
Total 18 17 01 25
Average 4.5 4.25 0.25 6.25
Range 04‐06 03‐06 01 25
5.3.3 Combined Analysis of RAPD and ISSR Markers
In the present study polymorphism level of conventionally grown wild type and in vitro propagated Aloe vera plants was investigated using RAPD and ISSR based molecular markers (Table 13). Tissue cultured plants produced maximum number of bands (32) with all RAPD primers and 18 bands with ISSR primers Wild type field grown Aloe vera plants produced 29 DNA bands with all primers of RAPD and 17 bands with
ISSR primers. Thus total 50 DNA bands of tissue culture plants were generated from all the 12 primers of both molecular markers, whereas total 46 bands of conventionally
144 Chapter 5 Molecular Analysis
grown plants were generated. In sum up of all the two molecular markers with both
Aloe vera plants total 94 bands were produced.
Table 13: Total number of scorable amplicons from RAPD and ISSR analysis
Molecular Markers Tissue culture Wild Total
RAPD 32 29 59
ISSR 18 17 35
Total 50 46 94
145
CHAPTER 6
GENERAL DISCUSSION
146 Chapter 6 General Discussion
6.1 GENERAL DISCUSSION
6.1.1 Acemannan Enhancement
The potential of plant cells as rich sources of commercially significant medicinal products is broadly acknowledged. Most cell cultures, however, have failed to produce or yielded very limited amounts of the expected natural substances. Many factors have been reported to have an effect on the synthesis of special plant products. They include nutrition, hormonal levels, light, temperature, pH, metabolic precursors.
Some researchers observed that the genetic variations observed during micropropagation can be influenced by the relative levels of cytokinins and auxins
(D’Amato, 1984). Likewise, in some studies it was proposed that the composition of the growth media play a strong role in influencing the genetic makeup of the plants subjected to micro-propagation. Recently, combined effects of all the above factors holding the potential to increase productivity were investigated for enhancing production of Aloe mannan in Aloe vera plant.
In this study the development of a callus route was availed to investigate the in vitro plants for enhanced production of the bioactive polysaccharide acemannan. The production media and light conditions played a vital role to increase the acemannan levels in stress conditions. The investigation revealed quite interesting results as the acemannan yield increases several fold in the in vitro regenerated callus cultures and
Aloe barbadensis plants. The formation of acemannan in cultured cells was induced by mannose and sucrose carbohydrates added to the culture media.
147 Chapter 6 General Discussion
Researchers have investigated that feeding carbon sources affected plant cell growth and biosynthesis of important metabolites. Significant research has been conducted in regard to the analysis of light versus dark, especially on the increasing effects of darkness on accumulation of soluble carbohydrates in plant cells. The existence & absence of light immensely impact the development and growth in plants. The plants that were grown in the darkness have shortened leaves, etiolated & show a decrease in the quantity of photosynthetic pigments (Taiz and Zeiger, 2010). And also the plants grown in absence of light don’t photosynthesize and hence don’t make carbohydrates.
The in vitro growth is mainly reliant on sugar included in the culture medium and the stocks available in the cultured tissues.
The causes of increase in sucrose concentrations on the substances of soluble carbohydrates & starch of Dendrobium shoots were investigated by Ferreira et al. in
2011 which were in vitro grown for 150 days in existence and absence of light.
According to his findings the upsurge in the sucrose concentration leads to the increase in the quantity of total soluble carbohydrates by almost six times greater in the dark than in light. According to Medina et al. 1998, in Medicago strasseri plant callus cultures induced from leaves reported higher carbohydrate content in darkness than those obtained in light conditions.
Mannans are widely spread amongst land plants and ManS and CSLA genes have been established in all land plants signifying that the mechanism for mannan biosynthesis exists in all land plants. As only small quantities of mannans exist in the walls of many angiosperms, this mechanism is turned off or functions at a very low level (Yen et al., 2009). Though, large amounts of mannans are gathered as storage
148 Chapter 6 General Discussion
carbohydrates in the seeds of many families of plant indicating that, during plant evolution this mechanism has been turned on various times independently.
Acemannan is a linear storage polysaccharide which comprises of mannose units located inside the protoplast of the cells. This polysaccharide is composed by β-
(1→4)-linked mannan partly acetylated in positions 2, 3 or 6 and few side-chains developed by galactose units connected to C6 (Manna & McAnalley, 1993; McAnalley,
1993).
Scientists have been working for decades to disentangle many regulatory networks that regulate the deposition of many cell wall polysaccharides. Thus, model systems have been proposed to understand galactomannan regulation and biosynthesis.
Developing fenugreek seeds are used as model systems for galactomannan synthesis as they are considered to accumulate large amounts of galactomannan in their endosperm. The analysis of expressed sequence tag’s (EST) profiling data indicates that Man enzyme from fenugreek endosperm specially used GDP-Mannose as substrate and its incorporation was 300 times higher than GDP-Glucose for galactomannan biosynthesis. Since mannose was used readily through its conversion to GDP mannose (Fry, 1988., Campbell and Reid, 1982).
Penelope and Caroline in 2002, studied galactomannan biosynthesis in protoplasts from carob. When protoplasts were fed with radioactive labeled D-mannose and glucose, there was higher incorporation in mannose as compared to that in glucose reflecting active galactomannan biosynthesis. Thus, protoplasts from developing carob endosperm retained the ability of their original plant to synthesize galactomannan.
149 Chapter 6 General Discussion
In conclusion, with the techniques used currently we are able to successfully improve the production of this medicinally potent compound acetylmannan. However, complete understanding of acemannan biosynthesis and enhancement on genetic level is a complex process and requires years of research and development. These findings may play a vital role in the improvement of the natural drug based pharmaceutical industry.
6.1.2 Future Prospects
Medicinal plants have played an important role in providing relieve to human sufferings. Whatever huge quantities they are in, the natural resources are still limited and will diminish eventually, therefore an effective strategy is required for their sustainable utilization. In this regard in order to meet the increasing demand of the resultant products, these plants must be cultivated in the required quantities.
Biotechnology has a tremendous contribution in the economy of Pakistan by genetically modifying the therapeutic and medicinal plants to boost the output of pharmacologically appealing natural components.
The methodology of organ and tissue culture is applied for fast growth and multiplication of plants, genetic perfection of harvests, procurement of disease-free emulations and clones and to preserve worth full germplasm (Bhojwani & Razdan,
1992). One of the main employments of plant tissue culture is swift reproduction or micro-propagation. Micro-propagation has superiority over customary and conventional propagation in enabling quick propagation in short time and space. While
Aloe barbadenesis spreads and reproduced involuntary in its natural state, but its propagation is too slow to make it viable for commercial production (Meyer & Staden,
1991). In addition plants grown under varied conditions will often produce different
150 Chapter 6 General Discussion
phytochemical profiles, or at least different quantities of the individual components
(Stevens et al., 2000; Taiz and Zeiger, 2006). Similarly, different cultivars within a species may also produce different levels of other bioactive components or other constituents which enhance/counteract their medicinal activities (Julinai et al., 2006).
The extraction procedure, treatment and handling of crude plant extracts may also affect the condition and therefore the bioactivity/efficacy of the phytochemical components. Due to the enormous therapeutic potential of Aloe vera there is a huge requirement for the Aloe plants. The ultimate objective of this research is to fulfill the demand by utilizing the techniques of in vitro micropropagation for the production of
Aloe on commercial scale economically and having identical, elite and disease free variety of Aloe vera plants of standard quality with high yields of bioactive compounds.
The above findings pave the way for further studies on biosynthesis of medicinally important plant metabolites, and efficient multiplication of large number of plants from selected cell lines bearing high yield of specific active ingredient. This can be exploited for genetic biotransformation studies and further breeding programs.
Consequently, intensified molecular and biochemical investigations on plant secondary pathways are needed to explore the future of plant cell cultures as producers of commercially useful compounds. The results of this research are very encouraging and will open up new vistas for future application and further advancement in plant biotechnology.
151
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PUBLICATION
176 Publication
Badar, Z., Khan, S., Saifullah, K. A., Musharraf, S. G., & Choudhary, M. I. 2014. In vitro and Biotransformational Studies of Aloe barbadenis Mill. Pak. J. Bot., 46(2): 679-
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177
APPENDIX
178
List of Tables
Table No Title Page No
Table 1: Showing the pharmaceutical activities of Aloe vera components 22
Table 2a: Preparation and Storage of Plant Growth Regulators 58
Basic ingredients of MS media employed in the micropropagation of Table 2b: 61 Aloe vera
Effect of BAP and NAA on callus formation in Aloe barbadensis after Table 3: 66 30 days of incubation under iIlumination
Effect of BAP and NAA on callus formation from explants in Aloe Table 4: 67 barbadensis, after 30 days of incubation under dark conditions
Effects of BAP and IBA on plantlet regeneration from calli in Aloe Table 5: 72 barbadensis
Table 6: Effect of Auxins on Root formation from Regenerated Plantlets 73
Table 7: Shows the wavenumber (cmֿ¹) of acemannan in Aloe species 95
-Shows the wavenumber (cmֿ¹) of acemannan in callus cultures and in Table 8: 95 vitro regenerated Aloe barbadensis plants from callus
Shows analysis of Acemannan concentration in conventionally Table 9: propagated Aloe barbadensis plant, in vitro callus cultures and 97 regenerated plant from calli under both light and dark conditions
Table 10a: Composition of master mix for RAPD-PCR reaction 133
Table 10b: Primer sequence used for the RAPD-PCR reactions 133
Total number of bands calculated from tissue cultured and wild type Table 11a: 140 Aloe vera plant with eight RAPD primers
Polymorphism percentage between tissue cultured and soil grown Aloe Table 11b: 141 vera plant by using 8 RAPD primers
Total number of bands calculated from Tissue culture and Wild type Table 12a: 143 Aloe vera plant with four ISSR primers
Polymorphism percentage between Tissue culture and Wild type Aloe Table 12b: 144 vera plant by using 4 ISSR primers
Table 13: Total number of scorable amplicons from RAPD and ISSR analysis 145
179
List of Figures
Figure No Title Page No
Figure 1 Aloe barbadensis plant 12
Callus formation from explants in Aloe barbadensis, (a) and (b) under Figure 2 68 illumination, (c) and (d) under dark condition
Plantlet regeneration from calli in Aloe barbadensis, (a), under Figure 3 73 illumination and (b) in dark conditions
Figure 4 Formation of roots in regenerated Aloe barbadensis, (a) and (b) 74
Structure of polydispersed β-1, 4-linked mannan substituted with O- Figure 5 77 acetyl groups (acetylated mannan; aloe polysaccharide/acemannan)
Figure 6 FT-IR spectrum of Aloe vera standard 94
Figure 7 FT-IR spectrum of Aloe vera plant gel 94
Figure 8 ¹H- NMR spectra of Aloe vera gel powder concentrate 200:1 99
Figure 9 ¹H-NMR spectra of Aloe vera standard 99
Figure 10 ¹H-NMR spectra of Aloe vera callus extract 100
RAPD profiles of Aloe vera wild type soil grown and tissue cultured Figure 11 plants with primers LC-76, LC-77, LC-81, LC-83, LC-87, LC-90, OPL-1 100 and OPQ-12
RAPD profiles of Aloe vera wild type soil grown and tissue cultured Figure 12 plants with primers LC-76, LC-77, LC-81, LC-83, LC-87, LC-90, OPL-1 138-139 and OPQ-12.
ISSR profiles of Aloe vera wild type soil grown and tissue cultured Figure 13 142 plants with primers ISSR-A, ISSR-G, ISSR-M and ISSR-O
180
List of Graphs
Graph No Title Page No
Graph 1 Shows the analysis of acemannan concentration in different Aloe 93 varieties
Graph 2 Shows the analysis of acemannan content in conventionally 98 propagated and in vitro regenerated Aloe vera plants
Graph 3 Shows the combined analysis of acemannan content under 101 carbohydrate stress conditions in Aloe vera calli and regenerated plant
Graph 4 Shows the analysis of acemannan content under sucrose stress 104 conditions in Aloe vera calli
Graph 5 Shows the analysis of acemannan content under mannose stress 104 conditions in Aloe vera calli
Graph 6 Shows the analysis of acemannan content under glucose stress 105 conditions in Aloe vera calli
Graph 7 Shows the analysis of acemannan content under galactose stress 105 conditions in Aloe vera calli
Graph 8 Shows the calibration curve of acemannan standard 106
Graph 9 HPLC-UV chromatograms of Aloe vera standard, samples and control 107-113 at λ 209 nm.
Graph 10 Growth curve analysis in the suspension culture of Aloe barbadensis 119
Biotransformation of compound 1 by Cell suspension culture of Aloe vii, xii Scheme 1 barbadensis 120
181