High frequency microcloning of Aloe vera and their true-to-type conformity by molecular cytogenetic assessment of two years old field growing regenerated plants
© Haque and Ghosh; licensee Springer. 2013
Received: 26 July 2013
Accepted: 11 October 2013
Published: 18 October 2013
Aloe vera (L.) Burm.f is an important industrial crop, which has enormous application in pharmaceutical, cosmetic and food industries. Thereby, the demand for quality planting material of A. vera is increasing worldwide. Micropropagation is the widely accepted practical application of plant biotechnology that has gained the status of a multibillion-dollar industry throughout the world and this techniques can be used to meet the industrial demand of A. vera. Present studies aim to develop a proficient methods of high-frequency true-to-type plantlet regeneration without intermediate callus phase for A. vera.
Nodal portion of rhizomatous stem of A. vera were cultured on Murashige and Skoog (MS) medium (Physiol. Plant. 15:473 – 497, 1962) supplemented with various cytokinin and A. vera leaf gel (AvG) as organic supplement. Number of proliferated shoots per explant was increased along with the regeneration cycles and on MS medium supplemented with 2.5 mg/L 6-benzylaminopurine and 10.0% (v/v) AvG, only 17.8 ± 0.35 shoots per explant were induced on 1st regeneration cycle whereas on 3rd regeneration cycle these number increase to 38.5 ± 0.44 shoots per explant on the same medium composition. AvG have an encouraging role to increase the proliferation rate and on 3rd regeneration cycle 27.6 ± 0.53 shoot per explant induced on 2.5 mg/L BAP, but these number increase to 38.5 ± 0.44 shoots per explant when 10.0% (v/v) AvG was added along with 2.5 mg/L BAP. After transfer of individual excised shoots to a one-third strength MS medium containing 20.0% (v/v) AvG, all the shoots formed whole plantlets with maximum number (9.6 ± 0.29) of roots per shoot. 95.0% of the regenerated plantlets survived on poly-green house. Normal flower appeared in 84.2% field growing micropropagated plants after 18 to 20 months of field transfer. Further, clonal fidelity of the two years old micropropagated plants was established by studying mitotic and meiotic chromosomal behavior and also considered the chromosome number and structural organization. There were no alterations in chromosome phenotypes, somatic haploid (pollen mitosis) and diploid chromosome count (n = 7; 2n = 14), or meiotic behavior. Randomly amplified polymorphic DNA analyses revealed there were no somaclonal variations among these regenerants.
These results confirm the very reliable method for large scale production of true-to-type plantlets of A. vera, which can be used for commercial purpose.
KeywordsAloe vera leaf gel Diploid and haploid karyotype Meiotic study Micropropagation, RAPD fingerprinting True-to-type regenerants
Aloe is an important commercial crop available in a wide range of species and varieties in international markets. A. vera has been used for medicinal purposes in several cultures of different countries: India, China, Japan, Greece, Egypt and Mexico for millennia (Marshall 1990). Different properties being attributed to the inner, colorless, leaf gel and to the exudate from the outer layers of Aloe leaf in a number of studies for several years (Reynolds and Dweck 1999 Ni et al. 2004; Liu et al. 2011). Due to the huge utilization in pharmaceutical, cosmetic and food industries (Vogler and Ernst 1999; Eshun and He 2004; Botes et al. 2008; Grace et al. 2008; Bedini et al. 2009; Rodríguez et al. 2010; Chen et al. 2012; Lad and Murthy 2013; Zapata et al. 2013), the demand for quality planting material of A. vera is increasing day-to-day. Mass propagation of uniform, healthy plants through tissue culture is the only viable technique for production of large numbers of clonal plants in a short time. Several attempt was taken for last few decades to develop tissue culture systems of Aloe spp. (Meyer and van Staden 1991; de Oliveira and Crocomo 2009; Singh et al. 2009; Das et al. 2010a; Gantait et al. 2011; Rathore et al. 2011b; Amoo et al. 2012 2013), but still the efficient regeneration protocols are requisite to large scale production of true-to-type plants of this commercially important species. Aim of our present studies is to develop a proficient and cost effective method for rapid and high frequency shoot multiplication and in vitro rooting of A. vera from rhizomatous stem explants. The genetic fidelity of micropropagation system needs to be ascertained before using it at commercial level (Goswami et al. 2013). Prior to the availability of DNA-based markers; cytological, morphological and agronomic traits were exploited for the selection of the superior genotypes. However, morphological markers are not considered reliable because they are affected by environmental and cultivation conditions. In latest studies, cytogenetic observation of micropropagated plants was investigated for the conformity of chromosomal change in structural or ploidy level (Das et al. 2010b; Rana et al. 2012; Das et al. 2013). Molecular markers are more powerful tools for studying genetic diversity and relationships between genotypes. RAPD fingerprinting can be used to trace genetic or epigenetic changes at the genome level (Arnholdt-Schmitt and Schaffer 2001; Leelambika and Sathyanarayana 2011). In recent years, RAPD based detection of genetic polymorphism have been found successful application in describing somaclonal variability/homogeneity of micropropagated individual of many plant species (Savita et al. 2012; Paridaa et al. 2013; Goswami et al. 2013; Cheruvathur et al. 2013; Kumar et al. 2013; Haque and Ghosh 2013a). Manipulation of the composition and ratio of plant growth regulators (PGRs) is often the primary empirical approach used for optimization of in vitro micropropagation methods (Shukla et al. 2012). The present study was thus aimed at the following: (1) induction and regeneration of plants via direct shoot regeneration, (2) RAPD profiles analysis and (3) comparative cytogenetic assessment of two years old micropropagated plants and mother plant.
Aloe vera (L.) Burm.f. plants growing in wild conditions were collected during September 2010 from Nallamalas ranges of the Eastern Ghats Mountains of the Andhra Pradesh state of India and maintained in our experimental garden. After removing all leaves, the rhizomatous stem were used as explant and washed with 2.0% (w/v) systematic fungicide (Thiram) for 25 min followed by 2.5% liquid detergent (Tween-20 solution) for 3 min and then surface-sterilized with freshly prepared 0.15% (w/v) aqueous solution of mercuric chloride (HgCl2) for 12 min and rinsed 3 times with sterile distilled water to remove traces of HgCl2. The explants (≈8 mm piece of rhizomatous stem from nodal portion containing axillary shoot bud) were cultured on MS (Murashige and Skoog 1962) basal medium containing 3.0% (w/v) sucrose and various concentration and combination of cytokinin [6-benzylaminopurine (BAP), Kinetin (KIN)] and Aloe vera leaf gel (AvG). For AvG preparations, mature fresh leaves of A. vera ware collected from experimental garden and kept half an hour to remove yellow liquid exudate, then washed thoroughly in running water. Then leaf skin was removed and the odorless, colorless mucilaginous leaf gel was peeled off with the help of stainless steel spoon and were homogenized in mixture-grinder. Then the homogenates were filtered with tea-net and this liquid was termed as ‘AvG’, which was stored at 4°C until use. AvG contains over 75 active ingredients (Hamman 2008) and serve as a nutritional supplement. The cultures were incubated in growth chamber maintained at 23 ± 2°C under a 16 h photoperiod with a photosynthetic photon flux density of approximately 50 μmol m-2 s-1 emitted from cool fluorescent tubes (Philips India Ltd.). At every 4 weeks intervals, the cultures were sub-cultured in their respective fresh media. After completion of every regeneration cycle (8 weeks), each individual shoots (≥ 2.0 cm) were separated from proliferated shoot clumps for in vitro rooting and then pre-existing explants were re-inoculated in their respective fresh media for next regeneration cycle.
Root induction of microshoots
Regenerated shoots (2.0-4.0 cm long) with 3-4 leaves were separated from clumps into single ones and were cultured on only agar-water medium (without any MS nutrients and sucrose) and three different strength of MS medium (full strength, two-third strength and one-third strength) supplemented with 3.0%, 2.0% and 1.0% sucrose respectively. Similarly, the effect of different concentrations of AvG (0%-40.0%) was also evaluated on rooting efficiency of microshoots.
Acclimatizing and field evaluation of regenerated plants
Rooted plantlets (about 6-8 cm) were transferred to small earthen pots containing ‘Soilrite’ (sterile, chemically inert horticultural graded perlite marketed by Keltech Energies Ltd., Bangalore, India) and covered with transparent polythene bags to maintain 90-99% relative humidity and were kept in 25 ± 2°C temperature and 16-h photoperiod for 25 to 30 days. Thereafter, the acclimatized plants were transplanted on earthen tubs containing a mixture of soil and vermin compost (3:1 ratio) and maintained inside the poly-green house (30 ± 2°C temperature and relative humidity of 60-65%) for another 3 months. Finally the plants were transferred to the field under full sunlight.
Mitotic karyotype study
In vivo mother plant and field grown two years old ex vitro micropropagated plants were used for cytological analysis. Total 25 root tips of mother plant as well as 125 root tips of 25 randomly selected micropropagated plants were excised, washed with tap water, and pre-treated with a saturated solution of Þ-dichlorobenzene for 4 h at 16-18°C. Pre-treated material was thoroughly washed with tap water, fixed in an ethanol/acetic acid solution (3:1; v/v) for 24 h at 4°C. For somatic chromosome counts and karyotypic analysis, fixed root tips were stained with 2.0% aceto-orcein: 1 (N) HCl (9:1 v/v) mixture followed by incubating for 2 h at room temperature. Then stained root tips were macerated and squashed in 45.0% acetic acid. Chromosome plates were observed in Leica DM750 microscope and photographed with Leica DFC295 camera. Minimum of 5 metaphase plates from each root tip were analyzed to determine the somatic chromosome number at the metaphase stage.
Meiosis & pollen mitosis study
For meiotic and pollen mitotic studies, young inflorescences were fixed at the appropriate stage in a fixative containing ethanol/acetic acid (3:1; v/v) for 24 h at 12-15°C. Smear preparations were made in 2.0% aceto-carmine following Sharma and Sharma’s (1980) methods. All the meiotic and pollen mitotic plates were observed in Leica DM750 microscope and photographed with Leica DFC295 camera.
Genomic DNA extraction
Genomic DNA was extracted from leaf tissue (excluding transparent gel like region) of both mother plant and 10 randomly selected field grown two years old micropropagated plants separately using CTAB protocol (Doyle and Doyle 1990) with slight modification. Fresh leaf tissue (≈100 mg) was grinded to powder in liquid nitrogen using mortar and pestle. Powdered tissue was placed in 1.0 ml of pre-warmed (65°C) extraction buffer (2.5% w/v CTAB, 1.5 M NaCl, 25 mM EDTA, 100 mM Tris HCl pH 8.0, 1.0% w/v polyvinylpyrrolidone) in a 1.5 ml microcentrifuge tube. Just prior to homogenization, 2.0 μl of β-mercaptoethanol was added to the tube and these were incubated at 65°C for 60 min. Immediately following homogenization centrifuged (1000 × g at 22°C) for 10 min and the supernatant was transferred to fresh 2.0 ml microcentrifuge tube. Then equal volume of chloroform: isoamyl alcohol (24:1 v/v) was added and mixture was gently mixed for 10 min by inverting the tube. Then centrifuged (1000 × g at 22°C) for 8 min to separate phases. The upper aqueous phase was transferred to a fresh microcentrifuge tube and repeats the chloroform isoamyl alcohol (24:1 v/v) step. DNA was precipitated with double volume of chilled ethanol for overnight at−20°C, then centrifuged (4,000 × g at 22°C) for 10 min. The pellet was air dried and re-suspended in 100.0 μl of Tris EDTA buffer. Then samples were treated with RNase at a final concentration of 50.0 ng/ml and incubated at 45°C for 60 min. Quality and quantity of DNA was monitored by spectrophotometry and gel inspection. Each sample was diluted at concentrations ranging from 45.0-55.0 ng/μl and stored at−20°C.
PCR were carried out in a total volume of 20.0 μl containing 50.0 ng of genomic DNA, 200 μM of the dNTP mix (Sigma), 1 X Taq buffer-A and 1 unit Taq DNA polymerase (GeNei™). All constituents except primer and DNA were prepared as 1 X master mix. Amplification was carried out in DNA Thermal Cycler (MJ Mini™, Bio-Rad). PCR used an initial denaturation of 94°C for 5 min, followed by 40 cycles of 94°C for 45 sec, 38-43°C for 60 sec and 72°C for 90 sec. A final extension step of 7 min at 72°C was included after the last cycle. A total 32 primers from OPA, OPC, OPG, OPJ, OPK, OPL, OPM, OPN, OPAC, OPAD, OPAE, OPAF series (Operon Technologies Inc, Alameda, USA) were used for amplification using the cycling conditions mentioned above. The amplified products (20.0 μl) were mixed with 4.0 μl of 6 X DNA loading dye (GeNei™) and were electrophoresed along with ‘100 bp Plus’ DNA ladder (Thermo Scientific) in a horizontal gel apparatus (PowerPack™ Basic, Bio-Rad) using 2.5% agarose gel (containing ethidium bromide) in 1 X Tris-acetate-EDTA buffer pH 8.0 at 60 Volt for 120 min. The gels were visualized and photographed using a Gel Documentation system (Gel Doc™ XR, Bio-Rad). All PCRs were repeated thrice to check their reproducibility. Only consistently reproducible, well resolved fragments were scored.
Each treatment contained three replicates with 10 explants per replicate. The data pertaining to the number of shoots or roots per explant were subjected to a one-way analysis of variance (ANOVA). The differences among the means were compared by high-range statistical domain using Duncan’s test with the standard software SPSS 16.0 version.
Results and discussion
Effect of PGRs and AvG on shoot regeneration
Effect of cytokinins and Aloe vera leaf gel (AvG) supplemented with MS basal medium on shoots regeneration of Aloe vera
MS medium + Supplement
Number of shoot (≥ 2.0 cm) per explant
Number of shoot (≥ 2.0 cm) per explant
0.0 ± 0.0a
1.0 ± 0.0a
8.3 ± 0.23c
16.2 ± 0.34c
14.5 ± 0.31f
27.6 ± 0.53h
11.7 ± 0.44e
23.1 ± 0.44f
5.2 ± 0.24b
12.8 ± 0.28b
8.2 ± 0.24c
17.7 ± 0.40d
9.7 ± 0.29d
20.3 ± 0.33e
Cytokinin (mg/L) + AvG (%)
BAP + AvG
2.5 + 5
15.9 ± 0.24g
33.3 ± 0.41j
2.5 + 10
17.8 ± 0.35h
38.5 ± 0.44k
2.5 + 15
14.3 ± 0.28f
30.9 ± 0.43i
2.5 + 20
12.5 ± 0.30e
24.3 ± 0.41g
During the initiation of culture the rhizomatous stem explants of A. vera exhibited excessive leaching of phenolic substances, a cause of browning of the culture medium when cultured on only cytokinin containing medium. But this problem was overcome when AvG supplemented along with BAP. According to earlier findings of Singh et al. (2009), incorporation of antioxidants (viz. citric acid, ascorbic acid, polyvinylpyrrolidone) to the culture medium promoted growth and prevented browning of the culture medium for A vera micropropagation. We know, along with the nutritional supplementary activity, AvG have strong antioxidant properties (Botes et al. 2008; Amoo et al. 20122013). Thereby, addition of AvG may serve antioxidants activities in the culture medium which not only minimized the browning of tissues but also reduced leaching of phenolic compounds, which is harmful for in vitro cultures.
Effect of AvG and nutritional strength of medium on root induction
Effect of the strength of MS medium and concentration of sucrose (S) and Aloe vera leaf gel (AvG) on in vitro rooting of Aloe vera (after 18 d of implantation)
Strength of MS medium, concentration of sucrose (w/v) and AvG (v/v)
Percentage of shoot showing root formation
Number of root per shoot [means ± SE]
Length of longest root per shoot (cm) [means ± SE]
Full MS + 3% S
3.3 ± 0.25a
1.5 ± 0.12a
Two third MS + 2% S
4.2 ± 0.23b
1.9 ± 0.08b
One third MS + 1% S
5.6 ± 0.26c
2.3 ± 0.08c
2.7 ± 0.33a
2.9 ± 0.14d
One third MS + 1% S + 10% AvG
7.3 ± 0.24d
2.4 ± 0.07c
One third MS + 1% S + 20% AvG
9.8 ± 0.29e
3.1 ± 0.10d
One third MS + 1% S + 30% AvG
9.2 ± 0.26e
2.8 ± 0.08d
One third MS + 1% S + 40% AvG
6.5 ± 0.23d
2.1 ± 0.10bc
Acclimatizing and field evaluation of regenerated plants
A total of 76 out of 80 (95.0%) in vitro rooted plantlets were successfully acclimatized for 25 to 30 days (Figure 1D). Thereafter, the acclimatized plants were transplanted on earthen tubs containing a mixture of soil and vermin compost (3:1 ratio) for next 3 months with 100% survival rate. Ultimately all plants were established in soil on field condition under full sunlight (Figure 1E). The majority of the micropropagation protocols do not deals with concern of the acclimatization process or they only mention that the acclimatization was tested with success, but we studied it thoroughly up to 2 years after acclimatizing. After 18 to 20 months of field transfer, 84.2% (64 out of 76) of the survived plants flowered normally (Figure 1F).
Diploid and haploid karyotype analysis
List of RAPD primers, their sequence, optimal annealing temperature (T m ) and banding pattern of both mother plant and field-grown micropropagated plants of Aloe vera
In conclusion, according to present protocol, high frequency of plantlets production was achieved without use of any auxin on any stage throughout the study, i.e. from explant inoculation to plantlet hardening, a totally auxin free culture system. The molecular cytogenetic evidence of the genetic stability and true-to-type conformity of the regenerants of this protocol make it valuable for large-scale propagation of Aloe vera at industrial level. So in this contexts present findings are totally innovative and unique as compare to previous studies.
SMH is working as a Research Fellow under the guidance of BG. BG is an Associate Professor in the Plant Biotechnology Laboratory, Department of Botany, Ramakrishna Mission Vivekananda Centenary College, Kolkata, India.
Diploid number of chromosomes
Aloe vera leaf gel
Cetyltrimethyl ammonium bromide
- MS medium:
Murashige and Skoog (Physiol. Plant. 15:473 – 497, 1962) basal medium
Haploid number of chromosomes
Polymerase chain reaction
Plant growth regulators
Randomly amplified polymorphic DNA.
SMH acknowledge to MOMA (Ministry of Minority Affairs) and UGC (University Grant Commission) for providing MANF (Maulana Azad National Fellowship). Both the authors are thankful to Swami Kamalasthananda, Principal, Ramakrishna Mission Vivekananda Centenary College, Rahara, Kolkata (India), for the facilities provided as well as his continuous enthusiastic encouragement for the present study. Also acknowledge DST-FIST program for infrastructural facilities.
- Amoo SO, Aremu AO, Van Staden J: In vitro plant regeneration, secondary metabolite production and antioxidant activity of micropropagated Aloe arborescens Mill. Plant Cell Tiss Organ Cult 2012, 111: 345–358.View ArticleGoogle Scholar
- Amoo SO, Aremu AO, Van Staden J: Shoot proliferation and rooting treatments influence secondary metabolite production and antioxidant activity in tissue culture-derived Aloe arborescens grown ex vitro. Plant Growth Regul 2013, 70: 115–122.View ArticleGoogle Scholar
- Arnholdt-Schmitt B, Schaffer S: Characterization of genome variation in tissue cultures by RAPD fingerprinting–a methodological comment. Plant Biosyst 2001, 135: 115–120.View ArticleGoogle Scholar
- Bairu MW, Aremu AO, Van Staden J: Somaclonal variation in plants: causes and detection methods. Plant Growth Regul 2011, 63: 147–173.View ArticleGoogle Scholar
- Bedini C, Caccia R, Triggiani D, Mazzucato A, Soressi GP, Tiezzi A: Micropropagation of Aloe arborescens Mill: a step towards efficient production of its valuable leaf extracts showing anti proliferative activity on murine myeloma cells. Plant Biosyst 2009,143(2):233–240.View ArticleGoogle Scholar
- Botes L, van der Westhuizen FH, Loots DT: Phytochemical contents and antioxidant capacities of two Aloe greatheadii var: Davyana extracts. Molecules 2008, 13: 2169–2180.View ArticlePubMedGoogle Scholar
- Chaudhuri AK, Chaudhury BR: Meiotic chromosome behavior and karyomorphology of Aloe vera (L.) Burm f. Chromosome Bot 2012, 7: 23–29.View ArticleGoogle Scholar
- Chen W, Wyk BEV, Vermaak I, Viljoen AM: Cape aloes–a review of the phytochemistry, pharmacology and commercialization of Aloe ferox . Phytochem Lett 2012, 5: 1–12.View ArticleGoogle Scholar
- Cheruvathur MK, Abraham J, Thomas TD: Plant regeneration through callus organogenesis and true-to-type conformity of plants by RAPD analysis in Desmodium gangeticum (Linn.) DC. Appl Biochem Biotechnol 2013, 169: 1799–1810.View ArticlePubMedGoogle Scholar
- Das A, Kesari V, Rangan L: Micropropagation and cytogenetic assessment of Zingiber species of Northeast India. Biotech 2013., 3: doi: 10.1007/s13205–012–0108-y doi: 10.1007/s13205-012-0108-yGoogle Scholar
- Das A, Mukherjee P, Ghorai A, Jha TB: Comparative karyomorphological analyses of in vitro and in vivo grown plants of Aloe vera L: BURM f. Nucleus 2010a, 53: 89–94.View ArticleGoogle Scholar
- Das A, Mukherjee P, Jha TB: High frequency micropropagation of Aloe vera L: Burm f as a low cost option towards commercialization. Plant Tiss Cult & Biotech 2010b,20(1):29–35.Google Scholar
- De Oliveira ET, Crocomo OJ: Large-scale micropropagation of Aloe vera . HortSci 2009,44(6):1675–1678.Google Scholar
- Doyle JJ, Doyle JL: Isolation of plant DNA from fresh tissue. Focus 1990, 12: 13–15.Google Scholar
- Eshun K, He Q: Aloe vera : a valuable ingredient for the food, pharmaceutical and cosmetic industries–a review. Crit Rev Food Sci Nutr 2004, 44: 91–96.View ArticlePubMedGoogle Scholar
- Gantait S, Mandal N, Das PK: In vitro accelerated mass propagation and ex vitro evaluation of Aloe vera L with aloin content and superoxide dismutase activity. Nat Prod Resh 2011,25(14):1370–1378.View ArticleGoogle Scholar
- Goswami K, Sharma R, Singh PK, Singh G: Micropropagation of seedless lemon ( Citrus limon L. cv. Kaghzi Kalan) and assessment of genetic fidelity of micropropagated plants using RAPD markers. Physiol Mol Biol Plants 2013, 19: 137–145.PubMed CentralView ArticlePubMedGoogle Scholar
- Grace OM, Simmon MSJ, Smith GF, Van Wyk AE: Therapeutic uses of Aloe L (Asphodelaceae) in Southern Africa. J Ethnopham 2008, 119: 604–614.View ArticleGoogle Scholar
- Hamman JH: Composition and applications of Aloe vera leaf gel. Molecules 2008, 13: 1599–1616.View ArticlePubMedGoogle Scholar
- Haque SM, Ghosh B: Field evaluation and genetic stability assessment of regenerated plants produced via direct shoot organogenesis from leaf explant of an endangered ‘Asthma Plant’ ( Tylophora indica ) along with their in vitro conservation. Natl Acad Sci Lett 2013a. doi: 10.1007/s40009–013–0161-z doi: 10.1007/s40009-013-0161-zGoogle Scholar
- Haque SM, Ghosh B: Micropropagation, in vitro flowering and cytological studies of Bacopa chamaedryoides , an ethno-medicinal plant. Env Exp Biol 2013b, 11: 59–68.Google Scholar
- Hashemabadi D, Kaviani B: Rapid micro-propagation of Aloe vera L via shoot multiplication. Afr J Biotech 2008,7(12):1899–1902.Google Scholar
- Kumar A, Prakash K, Sinha RK, Kumar N: In vitro plant propagation of Catharanthus roseus and assessment of genetic fidelity of micropropagated plants by RAPD marker assay. Appl Biochem Biotech 2013, 169: 894–900.View ArticleGoogle Scholar
- Lad VN, Murthy ZVP: Rheology of Aloe barbadensis Miller: a naturally available material of high therapeutic and nutrient value for food applications. J Food Eng 2013, 115: 279–284.View ArticleGoogle Scholar
- Lee M, Phillips RL: The chromosomal basis of somaclonal variation. Annu Rev Plant Physiol Plant Mol Biol 1988, 39: 413–437.View ArticleGoogle Scholar
- Leelambika M, Sathyanarayana N: Genetic characterization of Indian Mucuna (Leguminoceae) species using morphometric and RAPD approaches. Plant Biosyst 2011,145(4):786–797.View ArticleGoogle Scholar
- Liu X, Li J, Zhang Y, Li L, He D: Biological research advancement in Aloe . J Med Plants Res 2011, 5: 1046–1052.Google Scholar
- Marshall JM: Aloe vera gel: what is the evidence? Pharma J 1990, 24: 360–362.Google Scholar
- Meyer HJ, Van Staden J: Rapid in vitro propagation of Aloe barbadensis Mill. Plant Cell Tiss Organ Cult 1991,26(3):167–171.View ArticleGoogle Scholar
- Molnár Z, Virág E, Ördög V: Natural substances in tissue culture media of higher plants. Acta Biologica Szegediensis 2011,55(1):123–127.Google Scholar
- Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 1962, 15: 473–497.View ArticleGoogle Scholar
- Ni Y, Turner D, Yates KM, Tizard I: Isolation and characterization of structural components of Aloe vera L leaf pulp. Int Immunopharmacol 2004,4(14):1745–1755.View ArticlePubMedGoogle Scholar
- Paridaa R, Mohantya S, Nayak S: In vitro propagation of Hedychium coronarium Koen: through axillary bud proliferation. Plant Biosyst 2013. doi:10.1080/11263504.2012.748102 doi:10.1080/11263504.2012.748102Google Scholar
- Rana S, Dhar N, Bhat WW, Razdan S, Khan S, Dhar RS, Dutt P, Lattoo SK: A 12-deoxywithastramonolide-rich somaclonal variant in Withania somnifera (L.) Dunal–molecular cytogenetic analysis and significance as a chemotypic resource. In Vitro Cell & Dev Biol–Plant 2012, 48: 546–554.View ArticleGoogle Scholar
- Rathore MS, Chikara J, Mastan SG, Rahman H, Anand KGV, Shekhawat NS: Assessment of genetic stability and instability of tissue culture-propagated plantlets of Aloe vera L by RAPD and ISSR Markers. Appl Biochem Biotechnol 2011a, 165: 1356–1365.View ArticleGoogle Scholar
- Rathore MS, Chikara J, Shekhawat NS: Plantlet regeneration from callus cultures of selected genotype of Aloe vera L–an ancient plant for modern herbal industries. Appl Biochem Biotechnol 2011b, 163: 860–868.View ArticleGoogle Scholar
- Reynolds T, Dweck AC: Aloe vera leaf gel: a review update. J Ethnopham 1999, 68: 3–37.View ArticleGoogle Scholar
- Rodríguez ER, Martín JD, Romero CD: Aloe vera as a functional ingredient in foods. Crit Rev Food Sci Nutr 2010, 50: 305–326.View ArticleGoogle Scholar
- Samantaray S, Maiti S: Rapid plant regeneration and assessment of genetic fidelity of in vitro raised plants in Aloe barbadensis Mill: using RAPD markers. Acta Bot Gallica 2008,155(3):427–434.View ArticleGoogle Scholar
- Savita , Bhagat A, Pati PK, Virk GS, Nagpal A: An efficient micropropagation protocol for Citrus jambhiri Lush. and assessment of clonal fidelity employing anatomical studies and RAPD markers. In Vitro Cell & Dev Biol–Plant 2012, 48: 512–520.View ArticleGoogle Scholar
- Sharma AK, Sharma A: Chromosome techniques–theory and practice. 3rd edition. London: Butterworth-Heinemann Ltd; 1980.Google Scholar
- Shukla MR, Jones AMP, Sullivan JA, Liu CZ, Gosling S, Saxena PK: In vitro conservation of American elm ( Ulmus americana ): potential role of auxin metabolism in sustained plant proliferation. Can J Forest Res 2012, 42: 686–697.View ArticleGoogle Scholar
- Singh M, Rathore MS, Panwar D, Rathore JS, Dagla HR, Shekhawat NS: Micropropagation of selected genotype of Aloe vera L–an ancient plant for modern industry. J Sustain Forest 2009,28(8):935–950.View ArticleGoogle Scholar
- Vig BK: Spontaneous chromosome abnormalities in roots and pollen mother cells in Aloe vera L. Bull Torrey Bot Club 1968, 95: 89–95.View ArticleGoogle Scholar
- Vogler BK, Ernst E: Aloe vera : a systematic review of its clinical effectiveness. Brit J Gen Pract 1999, 49: 823–828.Google Scholar
- Zapata PJ, Navarro D, Guillén F, Castillo S, Martínez-Romero D, Valero D, Serrano M: Characterization of gels from different Aloe spp as antifungal treatment: Potential crops for industrial applications. Ind Crop Prod 2013, 42: 223–230.View ArticleGoogle Scholar
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