Development of photo-insensitive varieties in legumes would pave way to meet out nutritional security of developing countries.

1. “Classical and molecular breeding
approaches for developing photo insensitivity
in pulses”
S. PRIYANKA
ResearchScholar
TNAU,Coimbatore
GPB 891 - DOCTRAL SEMINAR I (0+1)

2. Breeding rule
• “ Even as the population doubled from three to six billion,
we managed to race ahead with all kinds of technological
and scientific events in agriculture - from using more
fertilizers to mechanization to advanced plant breeding."
- Nina Fedoroff
(Emeritus Professor of Biology)

3. Introduction
Photoperiodism
Phytochrome - The central mystery
Importance of photo insensitivenessin pulses
Breeding for photoinsensitivity
Screening methodologies
Case study
Varieties released
Conclusion

4. Photoperiodism
The plants require a certain day length for flowering i.e., the relative length
of day and night which is called as photoperiod
The response of plants to the photoperiodexpressedin the formof
flowering is called as photoperiodism
Garner and Allard
(1920)
“the relative lengthof the day is a factor of the firstimportance in the growth
and developmentof plants”
•(Biloxi )Soybeans
•(Maryland Mammoth) tobacco

5. Based on duration of the photoperiod:
 Short day plants - (16h Dark & 8h Light)
Eg - Soybean , Tobacco
 Long day plants - (14 -16 h light)
Eg - Sugarbeet, Spinach
Intermediate:
•Long Short Day Plants:
Bryophyllum.
•Short-Long Day Plants:
Certain varieties of wheat
and rye

6. Day neutral
Plants flower in all photoperiods ranging from 5 hours to 24 hours
continuous exposure
 tomato
 Cotton
 Sunflower
 cucumber
 certain varieties of peas & tobacco
Photoinsensitivity…
Variety which does not require a specific photoperiodfor flowering is called
photo – insensitive

7. Photoperiodic Induction
Plants may require one or more inductive cycles for flowering.
• An appropriate photoperiod in 24 hours cycle- one inductive cycle.
• If a plant which has received sufficient inductive cycles is subsequently
placed under un-favourable photoperiods, it will still flower.
• Flowering will also occur if a plant receives inductive cycles after intervals
of un-favourable photoperiods (i.e., discontinuous inductive cycles).
An increase in the number and continuous inductive cycles results in early
flowering of the plant (Eg: soybean)
Shagun Kandelwal

8. Photoperiodic stimulus is perceived by the leaves…..
Floral hormone is produced in the leaves which is then trans located to the
apical tip, subsequently causing the initiation of floral primordia.
cocklebur (Xanthium pennsylvanicum),a short day plant

9. Photoperiodic stimulus can be transmitted from one branch of
the plant to another branch

10. Grafting experiments in cocklebur plants have even proved that the
floral hormone can be trans located from one plant to another
“Florigen” – Macromolecule
This maypossibly be a RNA or
protein molecule which is trans
located from the leaf to the
apical tips (or meristems) via
phloem in photo-induced plants
(Corbesier and Coupland, 2005)

11. THE CENTRAL MYSTERY
“Molecular Biology has its central dogma
Flowering physiology we have two terminal dogmas and a central
mystery”
The dogmas are that one pigment, phyto-chrome, mediates the
initial photoperiodic reactions in all plants, and one hormone,
florigen, concludes them.
The central mystery is how, with a common beginning and a
common end, the intermediate reactions require darkness in
short day plants and light in long-day plants.
- L. T. EVANS
The lnduction of Flowering, 1969

12. Photosensitivity
Phytochrome
PR PFR Far red light
730-735 nm
Red light
660-665 nm
Inter-convertible
Successful purification of intact native phytochrome (oat
seedlings) was first reported by Vierstra and Quail in 1983
The native phytochrome is a soluble protein with a
molecular weight of about 250 kDa
Darkness

13. APOPROTEIN + CHROMOPHORE
• Each polypeptide has a prosthetic group called as chromophore
which is covalently linked to the polypeptide via a sulphur atom
(Thioether Linkage) in the cysteine residue of the polypeptide.
• The protein part of the phytochrome is called as apoprotein
• Apoprotein along with chromophore constitute holoprotein
None of the two components of phytochrome i.e., apoprotein and
chromophore, can absorb light alone.

14. PR & PFR - Cis and trans isomer

15. Occurrence of phytochrome
Angiosperms,gymnosperms,bryophytes and algae
• Phytochromes have directly been detected in different parts of seedlings,
in roots, cotyledons, hypocotyls, epicotyls, coleoptile, stems, petioles, leaf
blades, vegetative buds, floral receptacles, inflorescences, developing
fruits and seeds

16. TYPES OF PHYTOCHROMES
TYPE1 PHYTOCHROMES
 Predominate in etiolated seedling
 Encoded by PHY A gene
TYPE2 PHYTOCHROMES
 Green plant and seeds
 Encoded by PHY B, PHY C, PHY D & PHY E genes
Minor differences in molecular weight and spectral properties of
these two types of phytochromes

17. Phytochrome regulation (Arabidopsis)
(Eirini kaiserli & Joanne Chory, 2016)

18. (Eirini kaiserli & Joanne Chory, 2016)
Arabidopsis

19. Jung and muller; 2014
Arabidopsis

20. Status of pulse production, importand export
Ministry of Commerce& DES
Ministry of Agriculture& FarmersWelfare

21. Status of availability and production gap (2012-13 to 2014-15)
Source: Ministry of Commerce& DES,
Ministry of Agriculture & FarmersWelfare
To meet out the demand of Pulses, on an average 40 lakh tonnes of different pulses
has to be imported from other countries.
The major import share belongsto peas (39%) followed by lentil (17%), urd/mung
(16%), pigeonpea (13%) and chickpea (12%).
A negligible export 2.55 lakh tones on an average is also made with majorshare of
chickpea (94%)

22. Breeding for photo insensitivity…..
Strategies to overcome production gap
The cropping system approach to inculcate pulses under new niches
• chickpea in rice fallows;
• pigeon pea in rice-wheat cropping systems,
• rice bunds, high altitude /upland, spring/summer pulses
• inter-cropping and so on maybe the areas expansionstrategy.
-Directorate of Pulses development
Min. of Agri. & FW

23. Two forms:
Moderate height plant - early maturing, it is often grown as an annual
(Green pods)
Taller plant – Late maturing and is usually grown as a perennial. The dark
coloured pods contain 4 - 5 seeds
• Short-duration dwarf cultivars are usually day-length neutral
• Taller growing cultivars are usually short-day plants that will not readily
flower if day length is 12 hours or more.
Cajanus cajan

24. Breeding for earliness
- pigeonpea
• Traditional pigeonpea cultivars – 6 to 9 months to mature.
• Some early lines maturing in 120–130 days were bred, and they
succeeded in creating new production niches.
• Breeders at ICRISAT continued their efforts to reduce its maturity
further to help in widening its adaptation.
<50 days to flower,and their maturity is achieved in 80–90 days
-(Vales et al., 2012)
• Since in pigeonpea earliness is tightly linked to photo-insensitivity
(Wallis et al., 1981)
• Such cultivars can help in broadening the adaptation to warm season
windows (>20 °C) at higher altitudes (up to 1600 m), wider latitudes
(up to 40° N/S), and under short-fallow between two normal crops

25. Pigeonpea
Saxena et al; 2017

26. Adaptation of cajanus cajan (pigeon pea) in sub tropical Australia
-E.S. Wallis, P. C. Whiteman &D.E Byth
Photo sensitive varieties introduced – flowering duration of 100 days
Australia – limitation of early frost incidence
Early sowing :
Resulted in tall plants (difficulty in harvest) extended flowering period
( pest incidence – heliothis control was found to be difficult)
Selection of early flowering type
Short statured ,early flowering pigeonpea from ICRISAT line ICP7179
-Flowered rapidly in 52 to 60 days ( insensitive to photoperiod (16 h) at 28
0 S latitude)
Photo insensitivity Pest incidence

27. Cajanus platycarpus- potential source for photo
insensitivity
Cajanus platycarpus – tertiary gene pool
Many desirable traits:
• Extra- early flowering
• Photoperiodinsensitivity
• Prolificflowering
• Annuality
Barriers to hybridizationwas investigatedand found to be post-zygotic
Hybrids were produced by adapting embryo rescue techniques
- Mallikarjuna& Moss ; 1995

28. Molecular mapping of QTLs for plant type and
earliness traits in pigeon pea
Major constraints in pigeonpeaproductivity:
• Low harvest index, poor plant type, long crop duration varieties and
susceptibility of abiotic and biotic stress
Pigeopea genome size – 853 Mb (11 chromosomes)
• simple sequence repeat (SSR) and single nucleotide polymorphism (SNP)
markers (Indo-US Agricultural Knowledge Initiative) creating opportunities
for a large scale mapping of genes and quantitative trait loci (QTLs) for
important agronomic traits
- Kumawat et al
BMC GENETICS ; 2012

29. Mapping population - Pusa Dwarf/ HDM04-1
• Phenotypic evaluation of the plant type and earliness traits showed
significant variability in population
Based on the frequency distribution:
Days to flowering showed
bimodal distribution exhibiting
the involvement of major genes
governing it
F2:3

30. QTL – Days to flowering

31. • Pigeonpea has a large variation in the flowering and maturity time
Genetic mapping of these traits has direct implications for the
developmentof short duration high yielding pigeonpea varieties
• Two additive effect QTLs were identified for days to flowering
(qFL4.1 and qFL5.1) and alleles from the early flowering genotype
HDM04-1 at these loci decreased the time of flowering by 14.4
days and 5.54 days, respectively.

32. Gupta et al; 2014
Soybean – photo sensitive short day plant
4 maturity genes viz., E1,E3,E4 and E7 respond to photoperiod
(Tsubokura et al., 2013)
Dominantalleles – photo insensitivity and late maturity
While recessive alleles – photosensitivity and early maturity
•Genotypes adapting to higher altitudes – posses recessive alleles
on these loci making them photo insensitive to longer day conditions
•Equatorial regions (short day conditions)– posses dominant alleles
& photosensitive
Developing varietiesfor specificarea – identificationof
combinationof genes for concerned area and incorporating them
during breeding process

33. Polymorphicmarker for all photoperiodloci has been identified
6 photo insensitive genotypes and 21 varieties differing in photoperiodic
responses were used for SSR Polymorphism

34. Identification of polymorphic markersfor all photoperiodic genes would help in
marker assistedselection

35. Cluster analysis
• 27 genotypes were groupedinto five clusters
• Grouping based on origin,photo insensitivity,long juvenility and other
characters
• Cluster 1 & 2 – Indian genotypes
• Cluster 3, 4 & 5 -70 % of exotic germplasm
• 1 cluster – short day conditions
• Among indian genotypes,MACS 330 – photo-insensitive
Existence of photoperiodicgene based diversity suggests for initiating
breeding programmes to developgenotypes for differentareas

36. - Gupta et al., 2017
At presnt,10 major genes controlling flowering time and maturity
have been indicated in soybean
E1 and E2 (Bernard 1971)
E3 (Buzzell 1971)
E4 (Buzzell and Voldeng 1980)
E5 (McBlain and Bernard 1987)
E6 (Bonato and Vello 1999)
E7 (Cober and Voldeng 2001)
E8 (Cober et al. 2010)
E9 (Kong et al. 2014)
J (Ray et al. 1995).

37. Khan and Goyal; 2009
Two varieties of mungbean (Vigna radiata (L.) Wilczek)
K-851
PS-16
Treatments:
• 0.2% EMS
•0.02% SA (Sodium Azide)
•20 KR gamma ray

39. Screening methodologies
 Field planting at natural photoperiodconditions
-(Balogun et al., 2007)
 Controlledilluminationin vivo has been used to screen for
photosensitivity using indices such as
 Days to maturity
 Relative growthrates before and after flowering
 NEW APPROACH
Invitro screening procedure
- Rapid and cost effective

40. Invitro method – photo senstivity
Case study
In vitro screening - rapid and efficient
saves time, space and labor
Long photoperiods were reportedto enhance high chlorophyll contents in
cells,manifested by high greening level (Uozumi et al., 1993)
Compared genotypic variations in growth rate before and after flowering,
days to flowering and fibre yield (in vivo) and callus induction, formation of
green spots and embryogenic clusters (in vitro)

41. Differencesin greenness of callus of Kenaf genotypes
Tainung and V400
Photoperiodicresponse of kenaf genotypes:
The results obtained in vivo which utilized growth rate and maturity period
as indices matched in vitro results which utilized green spot formation by
callus cultures.
In both cases, Tainung1 was photosensitivewhile V400 was not.
Exploitation of
invitro procedure in
other crops would
highly benefit in
effective screening
for photo
insenstivity

42. COWPEA (Vigna unguiculata (L.) Walp.)
• Photo sensitive cultivars – early flowering and extremely dwarf in habit
when the day length is under 12.5 h (Ishiyaku & Singh; 2001)
• Association of photosensitivity with dwarfing is observed and its
controlled by single recessive gene
Inheritance of time to flowering in cowpea
Ishiyaku et al; 2004
Inheritance of time from sowing to flowering studied by crossing photo
insensitive genotype (Kanannnado) x photo sensitive genotype (IT97D-
941-1)
Exhibited quantitative inheritance
Additive and Additive X Dominance were predominant – time to flowering
Major 7 Gene pairs were found to control delay (6 days) in flowering

43. Urd bean (Blackgram)
• Development of short duration and photo insensitive genotypes
creates plants suitable for different cropping systems
Earliness and photo thermo sensitivity are recessive traits
(controlled by major genes)
- Sinha,1988
Summer season cultivated (photo insensitive)
 Pant U 19
 T9
 KM1
 TMV 1
-Gupta and Kumar; 2006

44. Identification of Photo-thermo Insensitive Wild
Accessions of Vigna
54 wild accessions (16 species) were screened
Two wild accessions of Vigna viz.,
V. glabrescens (IC 251372)
V. umbellate(IC 251442)
• photo-thermo insensitive
• Valuable resources for transferring this important trait into Vigna crops
Indian Institute of Pulse Research,Kanpur (2012)

45. Photo insensitive varieties- TNAU
Redgram:
Co(Rg)/CORG 9701
• Pure line selection of BB 9825
• 120-130 days
Lab lab:
CO 14
• CO 9 X CO 4
• 80 days
• extra early, compact type
CO 13
• CO 9 X Floriki field
• 120 days
• 10,000 kg / ha
CO 9 (Mutant) – Photo insensitive
Green gram :
CO 8 (COGG 923 X VC 6040A)
Blackgram:
TNAU CO6 (DU 2 X VP 20)

46. Photo insensitive varieties international scenario
*BARI – BANGLADESH AGRICULTURE RESEARCH INSTITUTE

47. BARImung-1
(1982): 1 t/ha
BARImung-2
(1987): 1.2 t/ha
BARImung-3
(1996): 1.3 t/ha
Mungbean varieties developed by PRC, BARI
BARImung-4
(1996): 1.4 t/ha
BARImung-5
(1997): 1.7 t/ha
BARImung-6
(2003): 1.8 t/ha
*BARI – BANGLADESH AGRICULTURE RESEARCH INSTITUTE

48. Reference:
Christian Jung and AndreasMuller, E. 2014. Flowering time control and applicationsin
plant breeding. Trendsin Plt. Sci.
Erini Kaiserli and Joannechory. 2016. The role of phytochromes in triggering plant
developmental transitions.In: eLS. John Wiley & Sons, Ltd: Chichester.