This case study investigated the genetics of aluminium tolerance in lentil. Two lentil genotypes (L-7903 and L-4602) showed tolerance based on root regrowth in a hydroponic assay with aluminium, while two other genotypes (BM-4 and L-4147) were sensitive. A staining assay also showed less aluminium accumulation in the tolerant genotypes. Segregation analysis of crosses between tolerant and sensitive genotypes found a 3:1 ratio in F2 and 1:1 ratio in backcrosses, indicating monogenic inheritance of aluminium tolerance.

1. Breeding for aluminium tolerance in crop plants
Presented by:
Vivek Kumar Singh
Admn. No. : 2013A42D
Date: 02/04/2016
Venue: Seminar Hall
Department of Genetics and Plant Breeding
COA, CCS HAU, HISAR
CREDIT SEMINAR (II)
ON

2. Introduction
• Soil acidity is one of the most important factors that affect crop production
worldwide.
• In the north-eastern region of India, more than 95% area is affected by soil
acidity.
• Among abiotic stresses limiting crop growth and productivity, aluminium (Al) is
certainly one of the most devastating environmental stresses which are the third
most abundant element in the earth curst after oxygen and silicon.
• Under low and high pH conditions (acidic and alkaline soils), Al dissolves in
various phytotoxic ionic forms, which affect productivity in crops.

3. • Toxic form of aluminium is absorbed by the roots, causing phytotoxicity,
primarily damaging the normal functioning of the roots, inhibiting their
growth by blocking the mechanisms of water and nutrients absorption
and transport.
• The reclamation of acidic soil through application of lime is an expensive
method, ineffective in the subsoil and in some cases heavy application
may have a deleterious effect on the soil structure.
• The best way of solving this problem is to develop aluminium-tolerant
crop cultivars with increased aluminium tolerance.

4. Aluminium Toxicity
Aluminium in soils is present as insoluble alumino-
silicates and oxides.
As the soil pH drops below 5, the hexahydrate
Al(H2O)6
3+, more commonly referred to as Al3+, is
solubilized into the soil solution.
This form of Al appears to be the most important
rhizotoxic Al species
(Kinraide, 1991)

5. The ionic form of aluminium (Al3+) in acidic soil condition is toxic to all living cells.
The ionic form of aluminium, inhibits:
Root growth: earliest Al-toxicity response
Cell wall: displacement of cations
Plasma membrane: interfere with protons involved in transport and signal
transduction
Cation uptake: inhibit the uptake of many cations including Ca2+, Mg2+, K+, and NH+
4
Al can bind to membranes of cellular organelles and can interfere with many metabolic
processes and cellular functions resulting in blockage of cell division in root tips, increases
the rigidity of DNA double helix by reducing DNA replication, reduces root respiration
(Kochian et al., 2005)

6. Genetic variability
• Natural genetic variation for aluminium tolerance in crops is well documented
(Foy, 1988)
Level of Al Tolerance Crop Plant Species
Highly Sensitive Barley, Durum Wheat, Lettuce, Pea
Sensitive Wheat, Oat
Moderately sensitive Triticale, Maize, Sorghum, Cabbage
Moderately Tolerant Rice, Rye
Tolerant Soybean, Pigeon pea
Highly Tolerant Tea, Buckwheat
Al-tolerance of Selected Crop Plant Species
Garcia-Oliveira et al., 2015

7. Mechanisms of Al-Tolerance
• Avoidance mechanisms that promote external detoxification of Al and
exclusion of Al
• Strategies adopted by plant species which prevent or restrict the Al-
uptake by roots
• Cell wall chemistry: formation of mucilage
: efflux of organic anions (Malate, Oxalate & citrate)
: secretion of phosphate
: secondary metabolites (phenolics)
A. Avoidance:

8. • Root mucilage is a gelatinous polysaccharide which is exuded from the
outer layers of the root cap
• Horst et al. (1982) found a higher Al sensitivity of cowpea roots without
mucilage.
• Phosphate (Pi) exudation could be an important Al exclusion mechanism
through the formation of Al-phosphate complexes
• Pellet et al. (1997) observed a constitutive phosphate exudation from
the root apex of Al tolerant wheat genotype Atlas66

9. B. Tolerance Mechanisms
• Internal detoxification of Al: allow plants to cope with Al once it
enters the root and shoot symplasm.
• Buckwheat & Hydrangea: ability to accumulate Al in their roots and
aerial parts
• These species detoxify internal Al by forming Al-organic acid
complexes and it seems that Al undergoes a ligand exchange with
oxalate and citrate
• Forest trees such as Pinus taeda adapted these both mechanisms to
protect them from Al-toxicity

10. Genetics of Al Tolerance
Crop Genes resistance References
Rice Monogenic Ferreira et al. (1999)
Polygenic Nguyen et al. (2001) & Khatiwada et al. (1996)
Wheat Monogenic Somers & Gustafson and Riede & Anderson (1995)
Polygenic Carver and Ownby (1995)
Maize Monogenic Rhue et al. (1978)
Polygenic Pandey et al. (1994)
Barley Monogenic Minella and Sorrells (1992)
Pea Monogenic Singh and Choudhary (2010)
Chickpea monogenic Singh and Raje (2011)
Soybean Polygenic Bianchi Hall et al. (2000)
Tomato Polygenic Singh et al., (2007)
Arabidopsis Polygenic Hoekenga et al. (2003)
Common bean Polygenic Araujo et al. (2002)

11. Gene Protein function Plant Species (References)
ALMT1 Malate transport efflux
Wheat (Sasaki et al., 2004); Arabidopsis (Hoekenga et al., 2006); Brassica napus
(Ligaba et al., 2006); Arabidopsis (Kobayashi et al., 2007); Rye (Collins et al., 2008)
ALMT1 Malate transport efflux Brassica napus (Ligaba et al., 2006)
MATE1/AACT1/Frdl4 Citrate transport efflux
Sorghum (Magalhaes et al., 2007); Barley (Furukawa et al., 2007); Arabidopsis
(Liu et al., 2009); Maize (Maron et al., 2010); Rye (Yokosho et al., 2010); Rice
(Yokosho et al., 2011); Wheat (Tovkach et al., 2013; Garcia-Oliveira et al., 2014)
MATE2 Citrate transport efflux Rye (Yokosho et al., 2010); Maize (Maron et al., 2010)
STOP1 C2H2- type Zn finger TF Arabidopsis (Iuchi et al., 2007); Wheat (Garcia-Oliveira et al., 2013)
ART1 C2H2- type Zn finger TF Rice (Yamaji et al., 2009)
STAR1 UDP-glucose transport Partial ABC protein Rice (Huang et al., 2009); Arabidopsis (Huang et al., 2010)
STAR2 UDP-glucose transport Partial ABC protein Rice (Huang et al., 2009)
ALS1 Partial ABC protein-function unclear Arabidopsis (Larsen et al., 2007), Rice (Huang et al., 2012)
ALS3 Partial ABC protein-function unclear Arabidopsis (Larsen et al., 2005)
Nrat1 Transporter specific for trivalent Al Rice (Xia et al., 2010)
MGT1 Mg uptake Transporter Rice (Chen et al., 2012)

12. Phenotyping for Al-Toxicity Tolerance
A. Hydroponics Assay
B. Pot Assay
C. Field Evaluation
D. Tandem Phenotyping Assay
E. In vitro screening

13. Hydroponic Assay
• Hydroponics allows non-destructive measurements of Al tolerance based
on root growth (Carver and Ownby, 1995)
• The screening is done by comparing root growth of seedlings in a pair of
hydroponic solutions with and without Al (Sasaki et al., 2004; Famoso et al., 2010)
• A number of histo-chemical assays such as:
Haematoxylin,
Eriochrome cyanine R,
Morin

14. Composition of Nutrient Media
Reagents Formula g mM M wt.
Potassium nitrate KNo3 3.29 0.65 101.11
Calcium chloride CaCl2.2H2O 2.94 0.40 147.02
Magnesium chloride MgCl2.6H2O 2.54 0.25 203.30
Ammonium nitrate NH4No3 0.16 0.04 80.04
Ammonium sulphate (NH4)So4 0.07 0.01 132.14
The most common nutrient solutions used in screening of cereals are
Magnavaca’s nutrient solution for maize, sorghum and wheat, and Yoshida’s
nutrient solutions for rice (Yoshida et al., 1976; Magnavaca et al., 1987; Magalhaes et al., 2004;
Sasaki et al., 2004 and Magalhaes et al., 2007).

15. Haematoxylin
• The haematoxylin assay is based on the
formation of coloured complex between
haematoxylin and the root-bounded Al (Polle
et al., 1978)
• Al treated seedlings will be stained in 0.2%
haematoxylin.
Non-stained = Tolerant (a)
Partially stained = Moderate (b)
Completely stained = Sensitive (c)

16. Eriochrome cyanine R
• Eriochrome cyanine R staining has been
extensively used for the measurement of root re-
growth
• If root apical meristem is irreversibly damaged,
the root tips remained intensively stained with
purple colour, whereas the part of the root which
grows after exposure to Al stress remains
unstained (Aniol, 1995)

17. Pot Assay
 Small pots either may be filled with pre-washed and air dried sands
soak with nutrient solution or may be filled with acid soils collected
from the target regions.
 This system is suitable for the analysis of aerial and sub-aerial plant
part characteristics.
 Root length, root dry-matter, shoot length, shoot dry-matter are
computed as tolerance index

18. Field Evaluation
• The evaluation of genotypes under field condition is imperative,
because the improved genotypes for Al tolerance will ultimately
grown in those areas which have major problem of Al toxicity.
• The other advantage of field evaluation is that large population can
be screened with relatively low cost and less efforts.

19. Tandem Phenotyping Assay
• A combination of hydroponic and soil assays seems to be the best
approach.
• The Al tolerance mechanisms identified in the hydroponic cultures
using histo-chemical assays could then be confirmed in the soil
bioassay
• Tandem phenotyping approach can be helpful for the better
understanding of both seedling as well as adult plant tolerance.

20. In vitro screening
Screening is done by evaluating callus development from different
genotypes under acidic medium containing various concentrations of
aluminium along with aluminium free acidic medium
(Deborah and Tesfaye, 2003; Dharmendra et al., 2011)
Alfalfa and sorghum are two of the few cases where an in vitro
technique found to successful in regenerating tolerant materials
(Conner and Meredith, 1985a; Parrott and Bouton,1990)

21. Breeding for Al Tolerance
• The choice of breeding methodology relies on a number of factors
such as the inheritance of the trait, genetic variability and the genetic
background of the trait
• Breeding methodologies such as backcross, pedigree, single seed
descent, doubled haploids and recurrent selection can be used to
develop improved cultivars .

22. Backcross Method
• Simplest breeding method for improving Al tolerance
• The large effect genes could be easily tracked through consecutive
backcross generations by histo-chemical markers at the early seedling
stage (Carver and Ownby, 1995)
• The transfer of major gene for Al tolerance using backcross method
has been performed in wheat (Carver and Ownby, 1995)
• Example: Carazinho to Egret,
Atlas66 to Chisholm and Century

23. Mutation Breeding
• Mutation treatment can also be used to rapidly increase genetic variability
for Al-tolerance for screening programmes.
• In barley, mutagenic treatment with N-methyl-N-nitroso urea (MNH) and
sodium azide yielded thirteen mutants with increased level of Al-tolerance
(Nawrot et al., 2001)
• EMS-mutagenized Al- sensitive Arabidopsis mutant, als3-1 could result in
seedlings that could sustain root growth in an Al-containing environment
that is highly toxic (Kelly et al., 2006)
• A number of genes such as ALS3, STOP1, ALS1 in Arabidopsis, and ART1,
STAR1, STAR2, Nrat1 and ALS1 in rice have been identified through
mutagenesis

24. Molecular Breeding
• Molecular markers for Al-tolerance have been applied in breeding programmes to
monitor expression of the desired alleles in different genetic background and also in
genetic diversity studies
• Markers associated with candidate genes TaALMT (wheat), HvMATE (barley), ZmMATE1
(maize) and SbMATE (sorghum) have been developed.
(Raman and Gustafson, 2011)
• Overexpression of TaALMT1 in transgenic barley had enhanced Phosphorus-use
efficiency (PUE) and improved grain yield.
(Delhaize et al., 2009)

25. Gene Source of gene Recipient Promoter
Relative root
growth
Proposed mechanism Reference
CS Rice Tobacco 35S 2.0-fold Citrate efflux Han et al., 2009
MDH Arabidopsis, E.coli Tobacco PrbcS 2.4-fold Malate efflux Wang et al., 2010
Pox Tobacco Arabidopsis 35S 1.7-fold Protect from oxidative stress Ezaki et al., 2000
ALMT1
wheat Arabidopsis 35S 4.0-fold Malate efflux Ryan et al., 2011
wheat Barley ubiquitin 20-fold Malate efflux Delhaize et al., 2004
MATE1/
AACT1/
Frd3
Barley Barley ubiquitin 2.0-fold Citrate efflux Zhou et al., 2013
Maize Barley ubiquitin 2.0-fold Citrate efflux Zhou, 2012
Sorghum Arabidopsis 35S 2.5-fold Citrate efflux Magalhaes et al., 2007
WAK1 Arabidopsis Arabidopsis 35S 3.0-fold Stress responsiveness Sivaguru et al., 2003

26. Tissue Culture
• Selection is earlier and faster in tissue culture than in the field.
• Several tolerant crop plants have been obtained from somatic callus
and microspore cultures (Karsai et al., 1994)
• Alfalfa, callus derived from acid-tolerant cultivars has been observed
to have greater ability to grow on acidified medium (Mezentsev et al., 1982)
• The tissue culture induced somaclonal variation is being used for
improving aluminium tolerance in rice, wheat, tomato and many
other crops.

27. CASE STUDY

28. Aluminium tolerance in lentil (Lens culinaris Medik.) with
monogenic inheritance pattern Singh et al., 2015, Plant Breeding
Objective: To determine genetics of Al tolerance
• Plant materials: L-7903, L-4602, BM-4 & L-4147
• Hydroponic assay: Aluminium tolerance was evaluated by the
protocol of Polle et al. (1978) with partial modifications by
haematoxylin staining of root apices in a nutrient solution culture.

29. Determination of aluminium accumulation by Morin stain:
• Morin, forms a highly specific complex with Al at acidic pH
• Al-stressed roots were stained with 100 µM Morin visualized using
fluorescence microscope
Results:
• On the basis of root regrowth the genotype: L-7903 & L-4602 had
long root regrowth (1.47 and 1.12 cm, respectively), compared to
sensitive parents BM-4 & L-4147 (0.27 and 0.33 cm, respectively)

30. Localization of Al by Morin stain:
• Morin, strongly binds Al, forming a complex that emits green
fluorescence.
• The intensity of Morin fluorescence was less in tolerant genotypes
than sensitive ones

31. • The segregation ratios obtained for Al tolerance and sensitivity in the
F2 and backcross generations were 3 : 1 and 1 : 1, respectively.
• Test of allelism confirmed the same gene was conferring Al tolerance
in both genotypes (L-7903 and L-4602) as the F1 was also tolerant and
no segregation of tolerant : sensitive was recorded.

32. Marker-assisted breeding for TaALMT1, a major gene conferring
aluminium tolerance to wheat
(Soto-Cerda et al., 2015, Biologia Plantarum)
Objective: to introgress the TaALMT1 gene from CAR3911 into the high
yielding Al-sensitive cv. Kumpa-INIA using a MAS-BC strategy.
Plant materials: Kumpa-INIA and CAR3911 both from the Wheat
Breeding Program of the National Institute of Agriculture Research
(INIA), Chile.
• Carazinho and ET8 genotypes (Al-tolerant), Chinese Spring (Al
intermediate), and ES8 (Al-sensitive) were used as controls
• Hydroponic assay: Aluminium tolerance was evaluated by the
protocol of Raman et al. (2005)

33. Results:
• RRE of CAR3911 and Carazinho were not inhibited at 5 μM Al and
only 20 % at 40 μM Al.
Introgression of Al-tolerance gene from CAR3911 into
Kumpa-INIA:

34. 70 BC1 lines
34 Al-tolerant 36 Al-sensitive
BC1-14 BC1-28
Kumpa-INIA X CAR3911
F1
X Kumpa-INIA,
Recombination events (WMC-331 & WMC-457)
Background selection using 151 polymorphic SSRs
72 % RPG 71.3 % RPG
58 Al-tolerant BC2 lines
BC2-14-10
86.7 % RPG
BC2-14-1
81.5 % RPG
X Kumpa-INIA
X Kumpa-INIA,
56 Al-tolerant BC3 lines
BC3-14-10-28
98 % RPG
Kumpa-INIA-TaALMT1
self
Aluminium tolerance
Hydroponically
5 μM Al
40 μM Al
98.2 % RRE
75.2 % RRE

35. Towards development of Al-toxicity tolerant lines in indica rice by exploiting
somaclonal variation
(Roy & Mandal, 2005, Euphytica)
Introducton:
• Tolerant plants were developed through in vitro screening of
embryogenic calli.
• The calli were derived from mature seed embryos and cultured on
medium stressed with different concentrations of Al2(SO4)3·18H2O.
Plant materials:
• Annada, Taichung Sen Yu, IR72 and S1P1 681032

36. Medium composition:
• Medium 1: MS supplemented with Al2(SO4)3·18H2O (30 and 60 ppm), 2,4-
D, NAA, and 0.4% Gelrite, pH 3.85
• Medium 2: MS fortified with 2,4-D, NAA, sucrose and agar, pH 5.8
• Medium 3: Identical to medium 1, except for the presence of reduced
amount of 2,4-D
• Medium 4: MS with BAP, kinetin, NAA, sucrose and agar. pH 5.8.
• The medium was used for plantlet regeneration.

37. 400 seeds
surface sterilized
Callus Induction Medium (Medium 1)
% Seed germination and callus induction recorded
sub-cultured (after 28 days)
Medium 2 (Callus Maintenance Medium with no Al stress)
sub-cultured (after 21 days)
Medium 3, with Al stress
Medium 2
sub-cultured (after 21 days)
Medium 4 (Regeneration Medium)
IR72 maximum regeneration

38. Conclusion
• Al-toxicity is a major crop production constraint associated with
strongly acidic soils of the world.
• Cereals are widely affected by Al-toxicity worldwide.
• Plant physiologists, developed fast screening methods using different
histo-chemical markers for the identification of Al resistant genotypes
at early seedling stages.
• Tandem phenotyping seems to the best approach.
• Development and use of Al-tolerant crop varieties is economically
feasible and eco-friendly.

39. • A number of genes for Al-tolerance have been identified in many
crops, like ALMT1 in wheat and MATE1 in sorghum and barley.
• A monogenic dominant Al tolerance gene, can be easily transferred
through backcross breeding technique.
• Development diagnostic markers associated with candidate genes
TaALMT, HvMATE, ZmMATE1 and SbMATE also helps enhance
efficiency of the conventional breeding.