Aloe vera- NUTRITIVE SUPPLEMENT IN TISSUE
CULTURE OF Aloe vera PLANTS
Aim
To study the nutritive properties of Aloe vera gel used in in vitro propagation of
shoot tip explants of A.vera plant.
Materials and methods
1.Plant material and explant disinfection
The A. vera plants (3 years old) were thoroughly cleaned. The shoot tip explants (0.5
cm height) were disinfected by soaking in pure alcohol for 30 s followed by 10%
sodium hypochlorite (15 min) and finally a solution containing 0.1% mercury
chloride (10 min). After these treatments, the remaining trace of the disinfectant
was removed.
2. Culture conditions and culture establishment
The basal medium used for culture induction was MS medium with 3% sucrose, 1
mg/L of (IBA), 0.1% rosemary essential oil and solidified by the addition of 0.6%
agar. The pH was adjusted to 5.8. The culture media and the used material were
sterilized for 20 min in an autoclave at a pressure of 1 bar. The cultures were placed
in an incubator under a 16/8 h of light/dark (36 µmole/m2/s of light intensity).
Explants were placed in test tubes (diameter 16 mm and length 100 mm) each
containing 20 mL of medium. The cultures were maintained at a temperature of 22
±2°C. The parameters measured after four weeks of culture were explant survival
rate, intensity of browning, infection rate, number of leaves per explants, and
average leaf length. All the experiments were implemented into ten replicates.
3. Shoot multiplication
Shoots multiplication trials have been conducted to determine the effect of the A.
vera leaf gel (AvG) on the regeneration of axillary shoots. For A. vera leaf gel
preparations, mature fresh leaves of A. vera were collected from the greenhouse
and kept for an hour to remove aloine exudate, then washed thoroughly in running
water.
The mucilaginous leaf gel was extracted, homogenized in mixture-grinder and then
filtered. The culture media used consist of the basic medium MS supplemented with
0.2 mg/L IBA and 3 mg/L BA with different concentrations (0; 25; 50; 100 g/L) of A.
vera leaf gel (AvG). The measured traits recorded after one month of culture were
the average number of axillary shoots and the average height of the axillary shoots.
4. Rooting of microshoots
For rooting tests, the axillary shoots measuring 2 -3 cm obtained during the
multiplication phase are placed on media with different strength of MS (Full MS, ½
MS, ¼ MS) and A. vera leaf gel (AvG) (0; 10; 20; 30 %) supplemented with 1 mg/L
IBA. After four weeks of culture, the rooting rate, the number of roots, and the
average length of the roots were observed.
5. Acclimatisation and soil transfer
Rooted plantlets were thoroughly washed and planted in plastic pots containing
soilrite and covered with transparent polythene bags to maintain humidity and were
kept in 25 -30°C temperature for 30 days.
The acclimatized plants were transferred to soil mixture of soil, sand and manure
(1:1:1, v/v/v) kept in the greenhouse. Finally, the plants were transferred to the field
under full sunlight.
6. Statistical analysis
Statistical analyses were performed with STATISTICA software 10. The one-way
analysis of variance (ANOVA) was performed to identify the effect of each
treatment. The Fisher’s least significant difference test at the 5% threshold was used
to compare means.
Results and discussion
The explants began to show the signs of shoot induction after 1 week of culturing.
All the induced shoots showed no signs of browning from the oxidation of phenolic
compounds. The average number of leaves per explant was 3.71 with an average
length of 2.94 cm.
It was found that when explants are treated with benzyaladenin (BA), the best
outcome of shoot induction percentage and average number of shoots per explant
was respectively 90% and 2.70 for 1.5 mg/L hormone concentration; whereas for
kinetin (Kn) it was 100% and 4.20 for 2 mg/L hormone concentration. However, the
percentage of success for the initiation phase reached 93.35% with 0.25 mg/L of α-
naphthalene acetic acid (NAA) and 1.5 mg/L of benzylaminopurine (BAP), the
average number of shoots per explant observed was 4.67. After successful initiation
of the culture (30 days after culturing), newly formed shoots were excised
individually from the induced explants and further cultured on MS supplemented
with 0.2 mg/L IBA and 3 mg/L BA with different concentrations of A. vera leaf gel
(AvG) to determine its effect on the regeneration of axillary shoots (Table 1).
The highest shoot multiplication was found on M3 containing MS media + 0.2 mg/L
IBA + 3 mg/L BA + 50 g/L AvG (13.27 ±5.11) followed by M4 : MS + 0.2 mg/L IBA + 3
mg/L BA + 100 g/L AvG (9.73 ±5.06). The M1 media (MS + 0.2 mg/L IBA + 3 mg/L BA)
and M2 (MS + 0.2 mg/L IBA + 3 mg/L BA + 25 g/L AvG) produced the lowest average
axillary shoots (5.00 ±2.27 and 6.40 ±3.46 shoots respectively.
Shoot elongation (2.50 ±0.89 cm) was found the longest on M2 medium (MS + 0.2
mg/L IBA + 3 mg/L BA + 25 g/L AvG) and the least shoot height was found in M4
(1.82 ±0.45). The control media M1 (MS + 0.2 mg/L IBA + 3 mg/L BA) was placed in a
group with M3 (MS + 0.2 mg/L IBA + 3 mg/L BA + 50 g/L AvG) with an estimated
average height of 2.27 ±0.94 and 2.21 ±0.84 respectively.
All three strength of MS medium (1, 1/2, ¼) with 0; 10; 20; and 30% of AvG resulted
in cent percent root induction (Table 2).
The maximum number of roots (5.93 ±1.39; 5.73 ±1.75) and the longest root (6.20
±1.32; 5.90 ± 1.43 cm) were recorded on control medium (Full MS) and 1/2 strength
MS medium supplemented with 10% AvG.
A higher number of roots per cultured shoot were obtained with AvG (10%)
treatment when compared with 1/2, 1/4 strength of MS medium with 20 and 30% of
AvG respectively. The lowest number and length of roots (3.73 ±1.62; 4.64 ±0.91cm
and 3.87 ±1.6; 3.77 ±0.59 cm) were obtained with 30% AvG treatment when
supplemented with 1/2 and 1/4 strength of MS medium respectively. So, the
addition of AvG to the medium not only increased the percentage of response and
number of root per shoot but also the growth of the plantlets was improved.
The plantlets were transferred pots containing garden soil, manure, and sand in the
proportion of 1:1:1, respectively placed under net. There was 100% survival rate.
Conclusion
Using the protocol described in this study, it is possible to improve tissue culture
protocol for in vitro propagation of A. vera through addition of A. vera leaf gel in the
culture medium as an alternative plant source of organic nutrients.
Reference
Aloe vera leaf gel, a new approach to enhance plant tissue culture by I.HAMDENI , S.
SLIM , A. BOULILA and T. BETTAIEB
Research Laboratory of Horticultural Sciences, National Agronomic Institute of
Tunisia, University of Carthage, Tunisia;
Research Unit of Biodiversity and Valorization of Resources in Mountainous Areas,
School of Higher Education in Agriculture of Mateur, University of Carthage, Tunisia;
Laboratory of Natural Substances, National Institute of Research and Physico-
chemical Analyses, Biotechpole of Sidi Thabet, Ariana, 2020, Tunisia.
Volume 82(4). Published in Journal of new sciences on April, 01, 2021.
www.jnsciences.org
E-ISSN 2286-5314
