The document discusses lucerne (alfalfa) breeding, detailing its progress, breeding methods, and constraints faced. It highlights advancements in breeding techniques over the past century and the development of genetically modified varieties, such as Roundup Ready alfalfa, while addressing the challenges associated with cross-pollination and genetic diversity. The future prospects emphasize the need for improved hardiness, disease resistance, and biotechnological approaches to enhance yield and adaptability.

1. TOPIC- "LUCERNE BREEDING: PROGRESS
AND CONSTRAINTS”
SUBMITTED TO :
Dr. D. P. Gohil
Course Teacher (GP. 511) &
Research Scientist (FC)
Main Forage Research Station,
Anand
SUBMITTED BY :
Boddu Sangavi
Reg : 2010120093
M.sc agriculture 1st semester
Genetics and plant breeding

2. SCIENTIFIC CLASSIFICATION :
Kingdom : Plantae
Subkingdom : Tracheobionta
Super division: Spermatophyta
Division : Magnoliophyta
Class : Magnoliopsida
Subclass : Rosidae
Order : Fabales
Family : Fabaceae
Genus : Medicago L.
Species : Medicago sativa L
 Common name: alfalfa,rijka
 Origin: south west asia
 Chromosome no.: 2n=4x=32[auto tetraploid]
 Wild relatives :Medicago falcata 2n=16 [diploid]
Medicago coerulea 2n=16 ,2n=32[diploid and tetraploid]

3. INTRODUCTION :
 It is also known as “queen of forage crops”
 Lucerne was most probably the 1st crop to be cultivated for hay
 The value of lucerne as feed for horses and other animals is as early as 490 bc
 It is derived primarily from M.corulea, a diploid 2n=16 that grows wild in grassland of
south west iran,caucasus regions and eastern Anatolia
 It is the most productive and nutritious forage crops grown for hay, silage and pasture
 Alfalfa plants are highly heterozygous and exhibit severe inbreeding depression
• Plant height: Errect and 2-3ft tall .
• plant have a woody crown at ground level
• Flower: purple in colour complete ,
• axillary and borne at the top of plant.
develop in dense clusters of 20-30 flowers around 12-15mm long.
• Pollination : cross pollination ,especially bees
• Out-crossing : > 80 %
• Pollen viability : 3-5 minutes

4. TRIPPING:
• Keel opens by force similar to release of a
spring under tension and pollen grains
disperse by explosive action .
• The release of the sexual column is called
‘tripping’the flower.
 The action of insects, primarily honey
bees foraging on the lucerne flowers
for honey and pollen, will often cause
this tripping to occur.

5. BREEDING METHODS:
 Introduction: Ladak Strain- cold resistance
 Mass selection
Other selection methods
a) S 1 selection
b) Top cross or half sib selection
c) Full sib selection
d)Open pollinated progeny test e.g. Grain Sask,
No. 666, Viking, Cossac, Buffalo, Anand-2, T-9, AL-3
 Production of Synthetic varieties : Moapa
a) Multiple Strain Varieties
b) Multiple clone varieties
 Production of Hybrids : Hybrid Force – 400
 Poly cross breeding - Lahonten, Vernal
 Marker assisted introgression
 Marker assisted selection
 Whole genome selection
 Inter specific hybridization
 Somatic hybridization
 Transgenic breeding

6. LUCERNE BREEDING : PROGRESS
The progress of lucerne breeding since past 10 decades have been through new
breeding approaches i.e., through evolutionary changes where new ideas in crop
breeding ,hybridization has been taken place which resulted in more than
10%genetic gain every year.
1940: DEVELOPMENT OF RANGER ALFALFA:
• Ranger alfalfa was developed by Dr. Tysdal of Nebraska through multiline
approach : 45 % Cossack strain, 45 % Turkstan strain, 10 % Ladak
• He recognized the importance of winter hardiness and bacterial wilt to forage
production developed
1953: VERNAL ALFALFA
• Dr. Brink of the University of Wisconsin developed vernal alfalfa.
• He made the first inter species cross of M. sativa x M. Falcata
• capture winter hardiness, and hybrid vigour.
1974: MAGNUM ALFALFA
• Dairy land Seed developed magnum alfalfa through the application of general
Combining ability for parental clone selections.

7. • Alfalfa breeding programs have focused on forage yield and quality, resistance to
biotic and abiotic stressors, and fall dormancy.
• Conventional breeding is typically based on simple phenotypic selection, in
which each individual plant must be phenotyped,
• and on pedigree-based methods which attempt to predict individual breeding
values based on pedigree.
• conventional selection – slow progress
• modern programs are turning to breeding techniques based on genotyping,
including marker-assisted selection (MAS) and genomic selection offer the
promise of more rapid breeding cycles and fewer necessary phenotypic
evaluations.
• In any such program, a training population of alfalfa must be genotyped for a set
of markers, and then the same population must be phenotyped for the trait(s) of
interest.

8. MASS SELECTION
 mass selection was the 1st type of genetic improvement undertaken.
 Eg: Grimm 451
 However, mass selection was not always effective in producing better varieties.
alfalfa breeders began using other breeding methods for the development of new
varieties.
RECURRENT SELECTION
• breeding of alfalfa is predominantly accomplished through recurrent phenotypic
selection methods because of economic limitations associated with hybridization in
this crop.
• the identification of individual plants with superior performance for a given trait or
group of traits.
• Once identified, the superior plants are intermated to produce a new population,
called F1 plants.
• The selection process then begins again on the improved population of plants.
• It usually takes 2–5 cycles of selection and reselection to exploit the genetic
variability present in the population.

9. POLYCROSS:
Collect a number of desirable plants and form a source nursery.
From the nursery 25 to 50 superior plants are selected
Grown in isolated nursery.
Random cross pollination takes place in the isolation.
The seeds are harvested
Harvested seeds are grown as progeny rows.
Then the best ones are selected and clonally propagated.
Selected clones are again raised in isolation for random crossing
Desired synthetic is established.

10. ADVANTAGES
readily propagated by vegetative cutings
as it is perennial,frequent replantings are not necessary
easier and cheaper to produce seeds than using
separate isolation blocks for top cross
it is possible to obtain new superior combinations by natural
crosses between selected clones in polycross nursery
Example of Poly cross progeny - Lahonten, Vernal

11. MARKER-ASSISTED INTROGRESSION:
DONOR STRAIN RECIPIENT STRAIN
 Genetic mapping is used to identify QTL
 requires between six and ten generations to obtain a population homozygous for the donor gene,
but with more than 95% of the recipient genome
 Using linked markers, the desired QTL allele is backcrossed into elite cultivars. If the QTL
allele has a large effect on the phenotype, then introgression will likely be usefully undertaken.
x
Desired gene

12. TWO MAIN HINDRANCES ARE:
The first impediment
• The initial identification of prospective QTL.
• Mapping in tetraploids is complex due to the presence of four homologous
chromosomes in each plant.
• Even though QTL may be detected, locating them precisely is quite difficult
• Precision will likely be low, particularly compared to a diploid map, ensuring that the
markers used for introgression are linked in coupling with the QTL allele of interest is
difficult.
The second impediment
• Arises as a direct result of the first, once markers have been identified that are
correctly linked to the right QTL allele, backcrossing needs to be done to several
individuals within the population of interest to maintain sufficient genetic variation to
avoid inbreeding depression.
• Not many QTL can be selected simultaneously in a population before segregation ratios
become unwieldy.

13. “MARKER-ASSISTED SELECTION”
• It identify markers linked to QTL and then use those markers for future breeding
• does not integrate well into recurrent selection schemes
used in essentially all current alfalfa breeding programs.
• If a QTL effect is large enough, then the marker-assisted
introgression approach works best
• Therefore, markers and QTL will not be easily fixed in a population.

14. WHOLE GENOME SELECTION
• It is a breeding technique wherein selection of individuals for breeding is made based on
genotyping many polymorphic sites (markers) using a statistical model that has linked these
markers to one or more desirable traits

15. • Wang et al. sequenced RNA from the shoots of 36 alfalfa accessions, along with
one each of M. sativa ssp. falcata and M. truncatula.
• The study found 54,278 unigenes, among which 4493 SSR markers were
discovered, and mapped the markers to the M. truncatula genome.
• A complete genome sequence for alfalfa has not yet been completed; hence the
reliance on the related M. truncatula genome as a reference for work in alfalfa.
• Compared with MAS, GS is better at identifying larger numbers of smaller-effect
loci and has produced promising results in many crop species
The application of markers in alfalfa, therefore, likely has more in common
with animal breeding methodologies in which whole genome selection holds the
most promise to improve genetic gain

16. GENOME SEQUENCING:
• Molecular genetics and breeding research hindered due to lack of a high quality reference
genome
The Chromosome-Level Genome Sequence of the Autotetraploid Alfalfa and Resequencing of
Core Germplasms Provide Genomic Resources for Alfalfa Research
-Chen Shen , Huilong Du ,ZhuoChen
• A chromosome-level haploid genome sequence for ‘Zhongmu No.1’ alfalfa, a heterozygous
autotetraploid of 816-Mb high-quality have been accomplished.
• The contig N50 is 3.92 Mb, and 49,165 genes are annotated in the genome.
• The alfalfa genome is estimated to have diverged from M. truncatula approximately 8 million
years ago.
 Genomic population analysis of 162 alfalfa accessions revealed high genetic diversity, weak
population structure, and extensive gene flow from wild to cultivated alfalfa.
 Studies showed that MsFTa2, a Flowering Locus T homolog, whose expression is up regulated
in salt-resistant germplasms, may be associated with fall dormancy and salt resistance.
 Taken together, these genomic resources will facilitate alfalfa genetic research and agronomic
improvement.

17. INTER SPECIFIC HYBRIDIZATION
• In the genus Medicago, interspecific crossing was reviewed previously (McCoy and Bingham
1988; Quiros and Bauchan 1988).
• Asymmetric M. sativa × M. arborea hybrids were also generated using cytoplasmic male
sterile M. Sativa as the female parent (Armour et al. 2008).
• Introgression of some of the M. arborea genome into M. sativa has been established, using
morphological and DNA markers for anthracnose disease resistance.
 Obtaining diploid and tetraploid
hybrids.
 PG-F9 and 12P are diploid plants that
produce a significant percentage of 2n
eggs and pollen, respectively.
 The PG-F9 × 12P cross produced 2x
and 4x hybrids.
 The typical greenish flower color
derives from crossing yellow-flowered
PG-F9 with purple-flowered 12P.

18. SOMATIC HYBRIDIZATION
• Somatic hybrids of M. sativa with the annual species M. rugosa and M. scutellata were obtained by
protoplast electrofusion (Mizukami et al. 2006).
• Introgression of M. rugosa chromatin into M. sativa chromosomes was demonstrated by GISH.
• Partial resistance to alfalfa weevil appears to have been transferred from M. rugosa M. sativa.
Morphology of somatic hybrids and their parents.
(a) Greenhouse grown somatic hybrids: M. sativa (+) M.
rugosa (left), and M. sativa (+) M. scutellata (right),
(b) Flower and leave of parent: M. rugosa (left), M. sativa
(centre) and M. scutellata (right),
(c) Flowers and leaves of M. sativa (+) M. rugosa somatic
hybrids,
(d) Flowers and leaves of M. sativa (+) M. scutellata somatic
hybrids
• This technique has not given practical results to date, but is still utilized to introgress traits from wild relatives.

19. GENETICALLY MODIFIED ALFALFA
• A genetically modified variety of alfalfa is Roundup Ready Alfalfa (RR Alfalfa) developed
by Forage Genetics using a gene construct owned by Monsanto in 2005
• In 1970, an organic molecule , glyphosate was discovered to be an effective broad-range
herbicide, able to control several different weeds.
• The GM variety of alfalfa has a single bacterial gene the EPSPS gene that was isolated
from Agrobacterium tumefaciens, strain CP4
• This gene codes for a C4-EPSPS enzyme instead of EPSPS (5-enolpyruvylshikimate-3-
phosphate synthase enzyme )
• EPSPS is involved in the production of aromatic amino acids necessary for plant growth
• Glyphosate interferes with the EPSPS enzyme in the shikimate pathway

20. Aromatic amino acids
Shikimate
pathway
EPSPS
S
Shikimate pathway
Shikimate
pathway
C4-EPSPS
R
GLYPHOSATE
GLYPHOSATE
TYR
TYR
TYR
RR Alfalfa
growth
unaffected
by
glyphosate
Aromatic
amino acids
Aromatic
amino acids
Lucerne growth
affected by
glyphosate
GM ALFALFA
ALFALFA

21. TRANSGENIC BREEDING - FOR INCREASING DRY MATTER
• Genetically modified M. sativa plants expressing TaMYB14 provide a viable option for
improving animal health and mitigating the negative environmental impacts of pastoral animal
production systems and reduced greenhouse gas emissions.
• TaMYB14 is present in pro anthocyanidins in the foliage but, Medicago sativa do not
contain PAs in leaves.
• From an R2R3-MYB transcription factor, TaMYB14, are identified from the foliar PAs -
accumulating legume Trifolium arvense and provide evidence that this transcription factor is
involved in the regulation of PAs biosynthesis in alfalfa plants constitutively
expressing TaMYB14 synthesized and accumulated PAs in leaves increase the yield up to
1.8% dry matter.
Visualised changes in flower colour in transgenic lucerne plants expressing TaMYB14 gene construct.

22. The most pressing concerns are:
• As alfalfa is perennial crop that is pollinated by bees ,GM
• contamination is inevitable
• The possibilities of gene flow, weed shifts, and weed resistance.
• Gene flow occurs when bees carry GM-alfalfa pollen to nonGM alfalfa on other
fields and results in the reduced genetic purity of that breed
• The M. truncatula genome sequence is nearly complete (Young and
Udvardi 2009), and will serve as the framework on which all alfalfa
genomics work will be built in the future.

23. ALFALFA HYBRID :
Hybrid Force-400 :
• The introduction of Hybri force-400 by Dairy land seed company utilizes a male sterility
system of hybridization called msSUNSTRA
• The world’s first hybrid released in 2001
• Increased vigor and stronger plants compared to non-hybrid varieties
• Plant greens up earlier and shows more rigorous re-growth
Hybrid Force-420/wet:
• Branched root trait.
• Aggressive forage yield capabilities
• High forage quality
• Distinctive rapid re-growth after harvest
• Solid persistence
• For low lying or poorly drained soils
Advantage over variety:
 Hybrid vigor is vivid 8 to 15% yield advantage.
 Hybrid plants are more resilient and can be harvested earlier
 providing improved forage quality with reduced risk of stand loss.
 Hybrid alfalfa recovers faster after cutting

24. CONSTRAINTS IN LUCERNE BREEDING:
• Lucerne is cross pollinated and makes it difficult to propagate and maintain
the identifying of lines due to heterozygous in nature
• It is self-incompatible
• It has small floral parts that makes difficulty in hybridization
• Since, most of them are perennials, it requires any years to evaluate the genotypes
• Degree of out crossing and combining ability of individual plants also differ depending
upon the interplant genetic variation on climatic condition and availability of pollinator.
• This situation make it difficult to select plants or clones with high combining ability
• Lucerne is intolerant to acidic soils, water logging and grazing intolerant
• The population of plants with high combining ability is usually small.
• Progress in increasing alfalfa forage yield has been minimal over the past 20 years. This
is due primarily to lack of pollen control in open pollinated synthetic varieties.
• All alfalfa varieties to date have been open pollinated synthetic varieties. New alfalfa
hybridization technology provides the tools to overcome the forage yield barriers that have
been hindering alfalfa breeder from progress
• Breeding methods for improvement of lucerne is more slowly evolved
• Selection of diploid genotypes at tetraploid level would be undesirable
• Additive genetic variance is more important among alfalfa plants selected for plant yield and
forage quality

25. Future prospects:
1) There is a need for the development of cold and drought hardy
Lucerne with degree of persistence for pasture and meadows.
2) To introduce genotypes from the iso-climatic regions and cross it with
locally adapted types to improve its genetic base and adaptive fitness over
wider areas besides improving forage yield potentialities of the crop.
3) Use of biotechnological approaches for creating genetic variability and its
utility in development of new varieties.
4) Adaptability of Lucerne would depend much on the achievements made
through indirect methods such as breeding for high seed production, stress
tolerance, diseases and pest resistance etc.
In the past 20 years 115 indigenous collection of lucerne have been restored in
NBPGR from Gujarat

26. USES :
• The most important characteristics of alfalfa is it's high nutritional quality as
animal feed.
• Alfalfa contains between 15 to 22% crude protein as well as an excellent source of
vitamins and minerals
• While the slender stems and sprouted seeds are sometimes eaten by humans,
alfalfa is mainly used as animal fodder.
• It is usually cultivated for hay, and is frequently used for silage or haylage,
dehydrated to make meal or pellets, or used fresh by grazing or cut-and-carry
• In addition to the traditional uses of alfalfa as an animal feed, alfalfa is beginning
to be used as a bio-fuel for the production of electricity, bioremediation of soils
with high levels of nitrogen

27. RESEARCH INSTITUTES RELATED TO FORAGE CROPS:
Indian grass land and fodder research institute – Jhansi
IGFRI has3 regional research stations:
• Southern Regional Research Station , Dharwad (Karnataka)
• Hilly Regional Research Station, Srinagar (J & K)
• Regional Research Station, Avikanagar (Rajasthan)
 AICRP on forage – IGFRI , Jhansi
• It has 22 coordinated centres all over the country
• Cultivated in Punjab, western districts of UP ,Maharashtra,Gujarat,tamil nadu and
west Bengal
• In Gujarat- Mehsana
Banaskantha
Kuttch
Sabarkantha developed as ecotypes for lucerne
Kheda
Bhavnagar

28. VARIETIES RELEASED
variety (CVRC-
Notification no
Developed through
and breeding
institution
Region
adoption
Characteristic/ Yield
(q/ha)
Chetak (S-
244)
441(E)
dated 21st August
1975)
through single plant
selection from local
material of Mathura
IGFRI, Jhansi
U.P. ,Punjab, Haryana,
Gujarat, M.P., Delhi
800-1000 (GF)
Tolerant to aphids
Sirsa Type
9
440(E) dated 21st
August 1975) -
Whole of
India
700-800(GF)
Sirsa-8 no. 13 dated 19th
December 1978) -
northern India
-
Type-9 no. 13 dated 19th
December 1978)
by mass selection
HAU, Hisar
northern India -
Co-1 19(E) dated
14th January
December 1982).
through mass selection from
Coimbatore local collections.
Taminlnadu and
Karnataka
600-800 (GF)
GAUL-1
(Anand-2)
596(E) dated 13th
August 1984)
through selection from
perennial type Lucerne
AAU, Anand
Gujarat,
Rajasthan and
Madhya
Pradesh
80–100 t/ha(GF)
GAUL-2
(SS-627)
- selection from Sirsa
material by GAU
AAU, Anand
Whole of
India
900-950(GF), bold seed, persistent.

29. LL
Composit
e 5
596(E)
dated 13th
August
1984
selection from 125
downy mildew
resistant clones
from Kutch
Punjab,
Hariyana
720(GF), 150(DM),
Highly resistant
LL
Composit
e 3
540(E)
dated 24th
July 1985
From twenty clones selected
from fast growing, high
yielding and downy mildew
resistant germplasm collected
from Gujrat state.
PAU, Ludhiana
Whole of India 790(GF), 188.00(DM) resistant to
Downy mildew frost,
and lodging
NDRI
Selection
No.1
- Selection from material from
Saurashtra and Kutch by
NDRI
- 100 t/ha(GF)
Anand-3 408(E)
dated 4th
May 1995)
AAU, Anand Gujarat, Maharastra,
Rajsthan, U.P.,
Hariyanaand M.P.,
900-1000 (GF)
Anand
Lucerne-3
(AL-3)
. 454 (E)
11th
February
2009)
at AAU, Anand through pure
line selection and population
improvement of the material
collected from Kutch area of
Gujrat
AAU, Anand
Gujarat 1000-1100 (GF)
Anand
Lucerne-4
2013 AAU, Anand North West zone
of India
Tolerant to lodging, no
shattering, responds to
recommended ecology
Anand-23 2013 AAU, Anand Punjab and
Rajasthan
Good Quality (>21%CrudeProtein),
Lower NDF (%) and ADF (%)and
higher IVDMD(%),Tolerant to Lodging,
No
shattering
Ref: IGFRI –forage crop varieties.
AICRP on forage crops

30. Thank you