Presentation on anther culture in vegetable crops

1. Anther culture
Pollen/microspore culture
Immature ovary culture
Immature ovule culture
1

2. Anther Culture
2

3. Outline of seminar
 Definition of anther culture
 History
 Principle of anther culture
 Haploids and their importance
 Androgenesis and its pathways
 In-vitro Procedure for Anther culture
 Factors influencing Anther culture
 Associated problems with Anther culture
 Experimental studies
 Conclusion
3

4. ANTHER CULTURE: It is the technique in which in vitro
culturing of anthers containing microspores or immature pollen
grains cultured on a nutrient medium for the purpose of generating
haploid plantlets.
4

5. Bergner 1921
First observed mature pollen grains can be
induced to proliferate in culture to form
haploid callus.
S Guha & Maheswari 1964
Direct development of embryos from
microspore by the culture of anther.
Look Back
Before……!
5

6. J P Bourgin & J P Nitsch 1967
Haploid Plantlets from Anther Culture
Nicotiana tabacum.
6

7. PRINCIPLE OF ANTHER CULTURE
•The production of haploid by plants exploiting the
totipotency of microspore.
•The normal development and function of the pollen cell to
become a male gamete is stopped and is diverted forcibly to
a new metabolic pathway for vegetative cell division.
7

8. Haploid plants contain a single
complete set of chromosomes.
8

9. Why there is need to develop haploids?
1. Development of homozygous lines
2. Fixation of heterosis
3. Mutational studies
9

10. Continued..
4. For production of biotic and abiotic stress resistant plants.
5. For cytogenetical research.
6. For induction of genetic variability at haploid level.
7. For evolutionary studies.
8. For genome mapping as genetic maps
10

11. Particulars Anther culture
Conventional
breeding
Time required for developing
pure lines
One year or one crop
season
6-7 years
Time required for developing
cultivars
3-5 years 8-10 years or more
Fixation of heterosis Possible Not possible
Expenditure/cost involve More Lesser
Identification of recessive
mutants
Very easy Difficult
Mapping population Permanent Temporary
Table 1. Comparison between anther culture and conventional
breeding
11

12. Androgenesis: is the process of induction and
regeneration of haploids and double haploids originating
from male gametic cells.
• Two types of androgenesis:
1) Direct androgeneis:- The microspore behaves like a
zygote and undergoes chance to form embryoid
which ultimately give rise to a plantlet.
2) Indirect androgenesis (Organogenic pathway):- The
microspores divide repeatedly to form a callus
tissue which differentiates into haploid plantlets.
12

13. • Pathway I
• Pathway II
• Pathway III
• Pathway IV
• Pathway V
Pathways for androgenesis
13

14. Requirements to trigger androgenesis
14
Healthy plant material
Ontogenesis
Temperature treatment

15. In-vitro Procedure for Anther culture
1. Collection of unopened flower buds
2. Surface sterilized with hot or cold
treatment
3. Anthers excised from flower buds and kept
separately
4. Anthers in first meiotic division is selected
by acetocarmine test
5. Inoculated in the medium containing
glutamine, L-serine and inositol
Contd…15

16. 6. Incubated the culture at 25 C for 15
days. Here, anthers grow in to embryoids.
7. Embryoids transfer to rooting medium
under 3000 lux illumination after 4-5
weeks the embryoids became plantlets.
8. After acclimatization, transfer to green
house
Cont…
16

17. 17
Fig. 1. Diagrammatic illustration showing various modes of androgenesis and
haploid plat formation by anther and isolated pollen culture.

18. Factors influencing Anther culture
18
1. Genotype of donor plants
2. Pollen development stage
3. Physiological state and growth condition of donor plant
4. Pretreatment
5. Surface sterilization before anther excision
6. Medium composition
7. Culture conditions

19. • Anthers fail to grow, embryos fail to continue its growth.
• Developing tissue or callus may be diploid or polyploid.
– Chimera of different ploidy may result.
• Low success rate - not commercially viable.
• Use of growth regulators for callus production usually
detrimental for haploid production since diploid and polyploid
cells are produced.
• Doubled haploids sometimes are not homozygous.
– Segregation may be seen in progeny.
Associated problems with Anther culture
19

20. Advantages of anther culture
20
 Simple.
 Less time consuming.
 Responsive.

21. Table 2. list of vegetable crops induced in-vitro androgenesis
Vegetable pathway Refrences
Asparagus A Pelletier et al., 1972 Dore 1974
Rutabaga A Machado et al., 1985
Black Mustard A Govil et al., 1989
Broccoli A Quazi 1978 Keller and Armstrong 1983
Brussels Sprout A Ockendon 1984, Lelu et al., 1985
White Cabbage A Kameya and Hinata 1970
Cauliflower A Ockendon et al., 1992
Chinese Kale A Kameya and Hinata 1970
Kale A Keller and Armstrong 1981, Kiefer et al., 1993
Sauerkraut Cabbage A Dore and Boulidard 1988
Petsai A Deng et al.,1982
Pakchoi A Zhong et al.,1978
Red Pepper A Wang et al., 1973
Sweet Pepper A George et al. 1973
Potato A Dunwell et al., 1973
Eggplant A Raina et al., 1973
Tomato A Gresshoff and Doy 1972
Watermelon A Xue et al., 1988
21

22. 22
Experimental
studies

23. Study - 1
23

24. Khatun et al.(2006) 24
Table 3. Effect of cultivars on callus induction
Cultivar Survaivability of
anther (%)
Days to callus
initiation
Callus induction %
Dohazari 21.66c 35.33b 18.33d
ISD-006 32.66a 28.41d 26.66a
Laffa- S 21.66c 38.41a 17.58d
Ishrudhi L 24.66b 31.66c 20.00c
IPM -31 19.58d 37.66a 15.33e
Jessore L 25.66b 28.75d 22.33b
LSD (0.05) 1.06 1.31 1.31
CV (%) 8.90 5.0.1 8.00

25. Khatun et al.(2006) 25
Table 4. Effect of media with different harmone supplementation
on callus induction.
Media with
different hormone
supplementation
Survaivability of
anther (%)
Days to callus
initiation
Callus induction %
MS + NAA 2mg /L
+ BAP 2mg/L
27.33a 32.20a 29.79a
MS + IAA 1mg /L
+ BAP 2mg/L
23.95b 32.83b 20.00b
MS + 2,4-D 2mg /L
+ BAP2mg/L
21.66c 32.54c 17.33c
LSD (0.05) 0.75 0.92 0.92
CV (%) 8.90 5.01 8.00

26. Khatun et al.(2006) 26
2

27. 25
Table 5. Effect of cultivars on root formation
Cultivars Days to root
initiation
No. of calli
showingroot
No. of root /callus at different week after
explanting
2nd 3rd 4th 5th 6th 7th
Dohazari 15.33a 1.25bc 0.33c 0.58c 1.19b 2.33bc 2.83b 3.50b
ISD-006 14.08b 2.25a 1.16a 2.00a 4.33a 4.66a 5.33a 5.66a
Laffa S 12.16c 0.66c 0.58bc 0.83bc 1.50bc 1.83c 2.58b 3.00b
Ishurdi L 13.00bc 1.16bc 0.66bc 1.16b 1.83bc 2.33bc 2.91b 3.58b
IPM 31 12.83bc 1.00bc 0.83ab 1.08b 1.83bc 2.41c 2.75b 3.25b
Jessore L 13.16 1.58b 0.58bc 1.16b 1.33c 2.16bc 2.75b 3.08b
LSD
(0.05)
1.17 0.64 0.37 0.46 0.52 0.51 0.59 0.54
CV (%) 10.64 9.92 6.45 9.99 10.21 13.90 8.74 10.02
27

28. Table 6. Effect of media with different hormone supplementation on root
formation.
Media
with
different
hormones
Days to
root
initiatio
n
No. of
calli
showing
root
No. of root /callus at different week after
explanting
2nd 3rd 4th 5th 6th 7th
MS + NAA
1mg /L +
BAP2mg/L
15.00a 0.87b 0.41b 0.87b 1.91b 2.29b 2.95a 3.45b
MS + NAA
2mg /L +
BAP2mg/L
13.04b 1.41a 0.75a 1.20a 2.04b 2.66a 3.25a 3.66a
b
MS + NAA
2.5mg /L +
BAP 2mg/L
12.25b 1.66a 0.91a 1.33a 2.41a 2.91a 3.37a 3.91a
LSD (0.05) 0.82 0.45 0.26 0.32 0.31 0.36 0.42 0.38
CV (%) 10.64 9.92 6.45 9.99 10.21 13.90 8.74 10.02
Khatun et al.(2006) 28

29. Khatun et al.(2006) 29
Fig. 3.

30. Khatun et al.(2006)
Fig. 4. Callus development from different media concentration
30
MS + NAA
2mg /L
+ BAP2mg/L
MS + IAA
1mg /L
+ BAP2mg/L
MS + NAA, 2.5mg
/L+BAP2mg/L.
Rooting media
MS + 2,4-D
2mg /L
+ BAP2mg/L

31. Anther culture in red cabbage (Brassica
oleraceae L. var. capitata
subvar. rubra): Embryogenesis and
plantlet initiation.
Cilingir et al., 2017, Turkey.
Study - 2
31

32. Table 7. The effects of media on callus induction (CI %), callus
maturation (CM %), embryo induction (EI %) and plantlet initiation (PI
plant/per callus)
Media CI (%) CM (%) EI (%) PI (%)
Sucrose
conc.
30g/l 100g/l 30g/l 100g/l 30g/l 100g/l
MS 52.0 A 56.0 A 40.0 B 30.0 A 7.5 BC 0.68 A 0.13 BC
Zencibas B5 26.0 B 40.0 B 20.0 C 12.5 B 0.0 D 0.58 A 0.0 D
Avg. 39 48.0 A 30.0 B 21.3 A 3.8 B 0.63 0.07
Different letters indicate a significant difference (P<0.05).
32

33. Figure 5.
(A)male flowers
(B)Microspores at uni-nucleate
stage
(C and D) Callus induction and
enlargement
(E) Callus maturation
(F-K) Embryogenesis
(L and M) Plantlets initiation
Cilingir et al., 2017.
33

34. Study - 3
34

35. Materials and methods
• Plant material: A cultivar of C. sativus L., Beta Alpha, and three
Iranian landraces of cucumber (Isfahani, Basmenj, and Korki)
were used in this experiment.
• Anther culture procedure.
-Experiment I: Effect of macronutrient concentration on callus
and embryo induction.
-Experiment II: Effect of agar concentration on callogenesis and
embryo induction and maturation.
• Cytological analysis.
• Statistical analysis.
35

36. Cultivar MN conc Anther culture efficiency
Callogenesis (%) EC % (ME/A) ± SE
Beta Alpha
0.5 50.00 f 10.00 e 0.13 ± 0.03 e
1 83.33 a–d 13.33 e 0.17 ± 0.03 e
1.5 90.00 a–c 13.33 e 0.17 ± 0.07 e
2 76.67 c–e 13.33 e 0.13 ± 0.03 e
Basmenj
0.5 83.33 a–d 10.00 e 0.13 ± 0.03 e
1 83.33 a–d 43.33 d 0.67 ± 0.12 b–d
1.5 90.00 a–c 70.00 ab 0.83 ± 0.03 a–d
2 70.00 de 43.33 d 0.50 ± 0.06 d
Isfahani
0.5 90.00 a–c 20.00 e 0.20 ± 0.06 e
1 96.67 ab 73.33 ab 1.03 ± 0.08 ab
1.5 100.00 a 83.33 a 1.26 ± 0.12 a
2 93.33 a–c 63.33 bc 1.23 ± 0.19 a
Korki
0.5 63.33 ef 13.33 e 0.13 ± 0.03 e
1 80.00 b–d 53.33 cd 0.93 ± 0.24 a–c
1.5 93.33 a–c 70.00 ab 1.00 ± 0.17 a–c
2 90.00 a–c 50.00 cd 0.63 ± 0.18 cd
Table8.Effect of macronutrient concentration on callus and embryo induction parameters in
anther culture of a cultivar and different landraces of cucumber (Cucumis sativus L.).
36

37. Figure 6(A). Effect
of agar
concentrations on
embryo maturation
percentage
Figure 6(B). Effect
of agar
concentrations on
mean number of
mature embryos
per each anther
Means followed by the same letter(s) indicate nonsignificant differences at P = 0.05
37

38. Figure 7. Callus induction and gametic embryogenesis in cultured
anthers of Cucumis sativus L.
38

39. Study - 4
Androgenesis induction, callogenesis,
regeneration and cytogenetic studies of
tomato haploid.
Farooq et al., 2010, Pakistan
39

40. Table 9. Effect of days to flowering on androgenesis.
Farooq et al., 2010, Pakistan
40

41. 55
Table 10. Effect of low temperature treatment on
callus initiation.
41

42. Table 11. Effect of sucrose concentration on
androgenic response.
Farooq etal.,2010 42

43. Study - 5
43

44. Materials and methods
• Anther-donor plants : C. annuum L. breeding lines (ATZ1, PO, TG, and
CDT)
- Intraspecific hybrids F1 (ATZ × PO), F1 (ATZ × TG), and F1 (ATZ ×
CDT)
• - DH lines derived from them (respectively AP40, AT4, andAC7)
- Species : C. frutescens L., C. chinense Jacq., and C. baccatum L. var.
pendulum
• - Interspecific hybrids : F1 (C. frutescens × C. chinense) and F1 (C.
frutescens × C. baccatum) and DH lines derived from them (FCH and
FA).
• Objective of experiment: To investigate the prolonged anther incubation
time with two different kinetin in R1 generation medium.
Olszewska et al., 2014 44

45. Table 12. The effect of anther incubation period on Culture medium
combined with kinetin in R1 medium on embryo formation.
45

46. Table 13. The effectiveness of regeneration and ploidy
level ofanther- derived plants.
46

47. Figure 8. Development of androgenic regenerants through in-vitro anther culture
of Capsicum spp. from anthers of AC7 DH line
Olszewska et al., 2014 47
A. Embryo emergence A. Acclimatized plant in GHA. Young plan for
acclimitization

48. 48

49. Conclusion
…
49

50. 50