Bridg, Hannia: Micropropagation and Determination of the in vitro Stability of Annona cherimola Mill. and Annona muricata L.

95

Chapter 7. Results
Random Amplified DNA Polymorphic (RAPD)

From the isolated A. cherimola and A. muricata DNA plant cell extract (Figure 26), the total concentration of DNA was determined fluorometrically. To test the quality of DNA extracted, an ethidium bromide agarose (1.5%) gel was run with 0.5 x TBE, 45 volts for 18 hours.

Figure 26. DNA extracted from A. cherimola and A.muricata

7.1 DNA Amplification

Genomic DNA of A. cherimola and A. muricata mother selections was amplified with 29 primers (Table 33).

174 scoreable bands yielded from the tested primers in A. cherimola and A. muricata mother plants established in the greenhouse (Table 33).

The number of RAPD bands scored for A. cherimola species was 45 compared with 42 bands for A. muricata species.

From the 29 primers only 6 produced clear amplification profiles, scorable and available for the DNA pattern band comparison in all the tested samples with A. cherimola selection Ch-A, Ch-Y, Ch-Z and A. muricata Mr-2, Mr-4, Mr-5. These primers were the C-5, C-11, Opa-16- Opa-18- Q-4 and Q-12 respectively.


96

Table 33. Arbitrary Primers used to screen A. cherimola and A. muricata by RAPD
(0) : no pattern, no repetition (1) : pattern and successful repetition

 

Primer

 

Sequence

 

A.cherimola

A. muricata

 

 

 

 

 

Ch-X

Ch-Y

Ch-Z

Mr-2

Mr-4

Mr- 5

 

 

 

 

 

 

 

 

 

 

 

1

B-11

5‘>

gTA gAC CCg T

<3‘

0

0

0

0

0

0

2

C-04

5‘>

CCg CAT CTA C

<3‘

1

1

0

1

1

1

3

C-05

5‘>

gAT gAC CgC C

<3‘

1

1

1

1

1

1

4

C-07

5‘>

gTC CCg ACg A

<3‘

0

0

1

1

1

1

5

C-11

5‘>

AAA gCT gCg g

<3‘

1

1

1

1

1

1

6

C-19

5‘>

gTT gCC AgC C

<3‘

0

1

0

1

0

0

7

G-19

5‘>

gTC Agg gCA A

<3‘

1

0

0

0

1

1

8

J-19

5‘>

ggA CAC CAC T

<3‘

1

1

0

0

0

0

9

L-04

5‘>

gAC TgC ACA C

<3‘

1

1

1

0

0

0

10

OPA-16

5‘>

AgC cAg CgA A

<3‘

1

1

1

1

1

1

11

OPA-17

5‘>

gAC CgC TTg T

<3‘

0

0

1

0

1

1

12

OPA-18

5‘>

Agg TgA Ccg T

<3‘

1

1

1

1

1

1

13

P-02

5‘>

TCg gCA CgC A

<3‘

0

1

1

0

0

0

14

P-05

5‘>

CCC Cgg TAA C

<3‘

1

1

1

1

0

0

15

P-10

5‘>

TCC CgC CTA C

<3‘

0

0

0

1

1

1

16

P-13

5‘>

ggA gTg CCT C

<3‘

0

0

0

0

0

0

17

P-20

5‘>

gAC CCT AgT C

<3‘

0

0

0

0

0

0

18

Q-04

5‘>

AgT gCg CTg A

<3‘

1

1

1

1

1

1

19

Q-05

5‘>

CCg CgT CTT g

<3‘

1

1

0

0

0

0

20

Q-06

5‘>

gAg CgC CTT g

<3‘

0

0

0

0

0

0

21

Q-07

5‘>

CCC CgA Tgg T

<3‘

0

1

0

1

0

0

22

Q-08

5‘>

CTC CAg Cgg A

<3‘

0

0

0

0

0

0

23

Q-09

5‘>

ggC TAA CCg A

<3‘

0

0

0

0

0

0

24

Q-10

5‘>

TgT gCC CgA A

<3‘

0

0

0

0

0

0

25

Q-11

5‘>

TCT CCg CAA C

<3‘

0

1

1

1

0

0

26

Q-12

5‘>

AgT Agg gCA C

<3‘

1

1

1

1

1

1

27

Q-13

5‘>

ggA gTg gAC A

<3‘

0

0

1

1

0

0

28

Q-14

5‘>

GgA CgC TTC A

<3‘

1

1

1

1

1

1

29

Q-17

5‘>

gAA gCC CTT g

<3‘

0

1

1

1

1

1

 

Total scoreable (1) bands per mother selection = 87

13

17

15

16

13

13

Total scoreable (1) bands= 174 with the 29 primers


97

7.2 Pattern Band Comparison

Five sets of PCRs were carried out with the 6 selected primers to improve the RAPD fingerprinting of each DNA sample from A. cherimola and A. muricata in vitro regenerants as well as from the mother source of explants or established Colombian and Chilean plants in the greenhouse (Table 34), (Figures 27,28,29,30).

The six tested markers Q-12, C-11, C-5, Q-4, OPA-18, OPA-16 showed monomorphic bands in A. cherimola (Figure 27; Figure 29) and A. muricata selections (Figure 28; Figure 30) when only the intensive bands were compared.

Table 34. Primers used in RAPD analysis and number of scoreable bands in in vitro clonal regenerants and mother plant of A. cherimola and A. muricata

The primers C-11, C-5 and Opa-18 in A. cherimola scored polymorphic bands on the var. Felpa, var. Bronceada and the Colombian selection. The primers Opa-18 and Opa-16 in A. muricata scored polymorphic bands between the Colombian selections (Table 16; Table 35). The mentioned primers register a variation between bands in terms of intensity, suggesting that they could be used to identify varieties or clone plants by RAPD.

Figure 27. Amplification of genomic DNA primers Q-12, C-11, C-5 primers from regenerants and mother plants of A. cherimola

bp = base pairs Lambda DNA/ EcoRI+HindIII marker
: muster band distinction

Figure 28. Amplification of genomic DNA primers Q-12, C-11, C-5 primers from regenerants and mother plants of A. muricata

bp = base pairs Lambda DNA/ EcoRI+HindIII marker

Figure 29. Amplification of genomic DNA primers Q-4, Opa-18, Opa-16 primers from regenerants and mother plants of A. cherimola

bp = base pairs Lambda DNA/ EcoRI+HindIII marker
: muster band distinction

Figure 30. Amplification of genomic DNA primers Q-4, Opa-18, Opa-16 primers from regenerants and mother plants of A. muricata

bp = base pairs Lambda DNA/ EcoRI+HindIII marker
: muster band distinction


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