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M.Sc. Biotechnology
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Aloe
Stemless
Native
It
In nature, A. vera is propagated through lateral buds which is slow, very expensive and
Hardening
Transfer to field
Assessment
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'
1.
DNA templates
1.0 L
2.
Primers
1.0L
3.
2.5 mM dNTPs
2.0L
4.
1.5 mM MgCl2
1.5L
5.
2.5L
6.
Sterilized D/W
16.5L
7.
Taq polymerase
0.5L
Initial Denaturation
2.
Denaturation
3.
Annealing
4.
Extension
5.
Final Extension
6.
Hold
4C for 5 min
Figure 1: Shoot regeneration from Shoot tip explants on standard regeneration media
Figure 2: Shoot multiplication after 16th day and 24th day of culture on
AM2(1.0mg/l BAP) medium respectively.
After 21 days the regenerated shoots were transferred onto media containing
different concentrations of auxin, data were recorded.
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 15 th
day of transplanting.
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.
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..
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.
Aggarwal D and Barna KS - Tissue culture propagation of elite plant of Aloe vera
Linn. J. Biochem. Biotech, 2004, 13: 77-79.
Bhandari AK, Negi JS, Bisht VK, Bharti MK- In Vitro propagation of Aloe vera, A
plant with medicinal properties. Nature and Science, 2010, 8(8):174-176.
Murashige T, Skoog F - A revised medium for rapid growth and bioassays with
tobacco tissue culture. Physiol Plant, 1962, 15: 473-479.
Namli S, Akbas F, Isikalan C, Tilkat EA, Basaran D - The effect of different plant
hormones (PGRs) on multiple shoots of Hypericum retusum Aucher. Plant Omics
Journal, 2010, 3 (1): 12-17.