Lucerne breeding - methods, progress and constraints

Lucerne is the most important fodder crop and it has specific tripping characteristic for pollination and it attained lot more progress and also through transgenic breeding and genetically modified herbicide resistant cultivars . It also has its own constraints . Uses of alfalfa

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 1 st 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 1 st 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 1 st 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. POLY CROSS:   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 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 i dentification 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 , g lyphosate 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: 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 Composite 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 Composite 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