possibility of lupinus angustifolius l. ovule …...the ovules were isolated from young disinfected...
TRANSCRIPT
Possibility of Lupinus angustifolius L. ovule development in in vitro cultures with applying wide crossing'
The research is carried out as part of the project no. 108 - Ministry of Agriculture and Rural Development
Renata Galek1, Anna Maciejewska-Hoza1, Ewa Sawicka-Sienkiewicz1, Bartosz Kozak1, Adela Adamus2, Agnieszka Kiełkowska2 Wroclaw University of Life and Environmental Sciences, Department of Genetics, Plant Breeding and Seed Production, Grunwaldzki 24a, 50-363 Wrocław
The aim of the study was to evaluate the development of the potential structures embryo sac of narrow-leafed lupin (2n = 40 chromosomes) after previous pollination using pollen grains originated from Lupinis mutabilis (2n = 48 chromosomes).
Four genotypes of narrow-leafed lupin were the objects of experiment: 'Graf', 'Emir', 'Karo' and line LAE-1. Presence on stigmas of L. mutabilis (two genotypes LM 13 and LM 34) pollen grains were checked. Germination of pollen grains tubes on stigmas and growing through the styles and
reached ovules after 48 and 96 hours after pollination were observed.
The ovules were isolated from young disinfected pods 7-10 days after pollination by L. mutabilis (LM13 or LM 34) and placed on two types of medium (P2_NLN, P3-ML6). Observations were performed after 7,
14, 21 and 30 days. The percent of ovules with different structure of tissue callus was founded. Development of three type of callus (I- needles, II - crystal, III hydrated) has been noticed.
Callus measurements - height (H) and width (W) have been specified.
Stigma of L. angustifolius after pollination
The presence of pollen grains on the stigmas and germination Narrow-leafed lupin pods - 4 days after pollination
Growing of the pollen grains tubes through the style and reached ovary
LM 34
’EMIR’
LM 13
GRAF
LM 13
LM 34
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
7 14 21 30
EMIRxLM13 P2_NLN EMIRxLM13 P3-ML6
EMIRxLM34 P2_NLN EMIRxLM34 P3-ML6
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
P2_NLN P3-ML6 P2_NLN P3-ML6
EMIRxLM13 EMIRxLM13 EMIRxLM34 EMIRxLM34
I II III
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
P2_NLN P3-ML6 P2_NLN P3-ML6
GRAFxLM13 GRAFxLM13 GRAFxLM34 GRAFxLM34
I II III
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
4,5
5,0
H I Width I H II Width II H III Width III
GRAFxLM13 GRAFxLM13 GRAFxLM34 GRAFxLM34
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
4,5
5,0
P2_NLN P3-ML6 P2_NLN P3-ML6
EMIRxLM13 EMIRxLM13 EMIRxLM34 EMIRxLM34
H I Width I H II Width II H III Width III
0,0
20,0
40,0
60,0
80,0
100,0
7 14 21 30
KAROxLM13 P2_NLN KAROxLM13 P3-ML6
KAROxLM34 P2_NLN KAROxLM34 P3-ML6
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
P2_NLN P3-ML6 P2_NLN P3-ML6
KAROxLM13 KAROxLM13 KAROxLM34 KAROxLM34
I II III
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
5
H I Width I H II Width II H III Width III
KAROxLM13 P2_NLN KAROxLM13 P3-ML6
KAROxLM34 P2_NLN KAROxLM34 P3-ML6
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
7 14 21 30
LAE1xLM13 P2_NLN LAE1xLM13 P3-ML6
LAE1xLM34 P2_NLN LAE1xLM34 P3-ML6
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
P2_NLN P3-ML6 P2_NLN P3-ML6
LAE1xLM13 LAE1xLM13 LAE1xLM34 LAE1xLM34
I II III
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
4,5
5,0
H I Width I H II Width II H III Width III
LAE1xLM13 P2_NLN LAE1xLM13 P3-ML6
LAE1xLM34 P2_NLN LAE1xLM34 P3-ML6
GYNOGENESIS
REASUME The use of wide crossing method has had positive influence on the development of pods. While isolated ovules showed induction of tissue callus. The ovules of L. angustifolius not treated with pollen of foreign species were characterised very rare regeneration. The LM34 pollen grains had a positive effect on the callus induction in the case of three analyzed forms of narrow-leafed lupin, especially in the Emir variety on both types of media. The weakest regeneration of callus tissue occurred in 'Graf'. Hydrated callus (type III) and crystal structure (type II) most often occurred in the analyzed explants. The results of the cytometric analysis indicated that in the most of regenerating ovules showed ploidy level 1x and 2x. Only in the case of regenerating ovules of the cv. Karo derived from wide crossing (LM13, on P2-NLN medium), the ploidy level was only 1 x. The research will be continued to analyze the changes in the embryo sac, as a result of the wide crossing and further regeneration of callus tissue as well development of plants.
Results of flow cytometry A – 1x, B, 2x
I- needles
Figures representing % of ovules with callus (7, 14, 21 and 30 days)
Figures representing % of type callus structures
Figures representing sizes of callus
-
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
7 14 21 30
GRAFxLM13 P2_NLN GRAFxLM13 P3-ML6
GRAFxLM34 P2_NLN GRAFxLM34 P3-ML6
Regeneration of ovules after wide crossing
II - crystal III hydrated
Pods before isolation of ovules Pods before isolation of ovules