effect of growth regulators and genetic fidelity testing of aloevera

1. By:-
Smriti Singh
M.Sc. Biotechnology

2. •Member of the lily family.
•Around 300 species of Aloe.
•Aloe Vera, Aloe Barbadensis.
•Stemless or short stemmed succulent plant.
•Native to Africa, but is now widespread.
•It is a bitter herb with a wide range of medicinal
properties.
•It contains over 75 compounds, many of which are
biologically active.
•Out of the 22 amino acids required for the human body, 8
essential amino acids are found in Aloe vera.

3.  In nature, A. vera is propagated through lateral buds which is slow, very expensive and
low income practice.
 Providing quality planting material to farmers on commercial scale by traditional
propagating method is not possible due to 60 per cent sterility and low rate of
propagation.
 Seeds take around 1-6 months for germination.
Therefore, there is a need to develop suitable and alternative method for
traditional propagation like in vitro propagation including tissue and organ culture is
used for rapid multiplication of plants for genetic improvement of crops, for obtaining
disease-free clones and for progressive valuable germplasm.
 In-vivo regeneration is too slow and insufficient to meet the industrial demand.
Mass propagation through in vitro micropropagation gives an attractive
strategy to fulfill the commercial demand for planting material.

4.  Establishment of shoot culture.
 To study the effect of different growth regulators on in-vitro
multiplication of Aloe vera.
 To study the effect of different growth regulators on in-vitro rooting of
Aloe vera.
 Genetic fidelity testing of in-vitro raised plants.

5.  In vitro propagation of Aloevera
 Running healthy suckers of Aloevera were used as explants which were
taken from CPB HISSAR.
 Inoculation on establishment media.
 Transfer to the MS media supplemented with different concentrations of
BAP, KIN, IAA, NAA and IBA which were used for shoot multiplication.
 After 21 days the regenerated shoots were transferred onto media
containing
different concentrations of auxins for root induction.
 Hardening
 Transfer to field

6.  Assessment of Genetic Fidelity testing
For this purpose, 6 in vitro raised, hardened plants(A1, A2, A3, A4, A5 and A6) were
chosen randomly from the population and compared with the mother plant (M) from
which the explants were taken.
DNA Isolation
For DNA isolation CTAB method was used
Table 1: RAPD primers used for studying Genetic Fidelity in Aloevera
S.No Primer Sequence
1. OPA-08 5'<GTGACGTAGG>3'
2. OPC-16 5'<CACACTCCAG>3'
3. OPD-05 5'<TGAGCGGACA>3'
4. OPL-18 5'<ACCACCCACC>3'
5. OPN-3 5'<GGTGCACGTT>3'

7. 1. Initial Denaturation 94°C for 5 min.
2. Denaturation 94°C for 30 sec.
3. Annealing 35°C for 1 min., 36 cycles
4. Extension 72°C for 2 min.
5. Final Extension 72°C for 5 min.
6. Hold 4°C for 5 min
1. DNA templates 1.0 µL
2. Primers 1.0µL
3. 2.5 mM dNTPs 2.0µL
4. 1.5 mM MgCl2
1.5µL
5. 10X PCR buffer 2.5µL
6. Sterilized D/W 16.5µL
7. Taq polymerase 0.5µL
Table 3: Reactions were run with the following cycling conditions:

8. • Surface sterilization of explants
• Establishment of aseptic culture :
Data for percentage survival and contamination were recorded after 21st
day of inoculation.
On 21st
day of inoculation 83% survival and 17 % contamination was reported.
Figure 1: Shoot regeneration from Shoot tip explants on standard regeneration media

9. The regenerated explants were further cultured on standard multiplication media
{MS + BAP (1.0mg/l) + NAA (0.5mg/l)}. After sufficient multiplication the
shoots were transferred on medium fortified with different concentration of
growth regulators and data were recorded for number of shoots after fixed interval
of time i.e. 7th
, 14th
, 21st
day of inoculation.
Figure 2: Shoot multiplication after 16th day and 24th day of culture on
AM2(1.0mg/l BAP) medium respectively.

10. Table 4: Effect of different growth regulators on in vitro multiplication of Aloe vera
(number of shoots)

11. After 21 days the regenerated shoots were transferred onto media containing
different concentrations of auxin, data were recorded.
Figure 3: Root induction from regenerated shoots after 15th day

12. Table 5: Effect of different growth regulators on in vitro rooting of Aloe vera

13. The well rooted plantlets of Aloe vera were separated from the culture bottles,
washed and transferred to polybags containing mixture of sand + soil +
vermicompost in 3:1:1 for hardening. The 100% survival was recorded after 15th
day of transplanting.
Figure 4: Hardened plants of Aloe vera on 15th day of transplanting.

14. • A better analysis of genetic stability of plantlets can be made by using DNA markers which
amplify different regions of the genome. Hence, in the present study, PCR based techniques;
RAPD was adopted for evaluation of clonal fidelity of Aloe vera plantlets.
Figure 5: Agarose gel electrophoresis of RAPD fragments of in vitro regenerated plantlets(A1-A6)
from mother plant (M) showing monomorphic bands generated by primer OPA-08

15. In our present study the number of scorable - bands for each RAPD primers are same
from sample1 (M) to sample 7(A6). The 2 RAPD primers generated a unique set of
amplification products. No polymorphism was detected during the RAPD analysis of
in-vitro raised plantlets of Aloe vera L..

16. The present study is aimed to test the “Effect of different growth regulators on in vitro
propagation and Testing of genetic fidelity of in vitro raised plantlets of Aloe vera L.”
The findings of the experimentation achieved during the present investigation are
summarized as under:
 The running suckers were used as explants for in vitro propagation of Aloe vera.
 The survival percentage of explants was obtained 83% when the explants were surface
sterilized with 0.2% bavistin & 0.2% streptocyclin (45 minutes) and 0.1% mercuric
chloride (4 minutes).
 Total of 22 different combinations of MS media with BAP, KIN, NAA, IAA and IBA
were used for in vitro multiplication of Aloevera.
 Maximum shoot multiplication response was observed (11.6 shoots) on MS basal
medium supplemented with 1.0mg/l BAP. Results of shoot multiplication concluded
that it is better to use BAP instead of KIN.
 Average no. of days required for was 8 days on half strength MS basal medium
supplemented with 1.5 mg/l NAA. Results concluded that NAA is better and more
effective for rooting.
 Survival rate of regenerated plantlets transferred to potting mixture containing sand +
soil + vermicompost (3:1:1) was 100%.
 Genetic fidelity testing showed that in-vitro raised plants and mother plant from where
explants have been taken are true to the type.

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