**3. Advances in improving reproductive ecology of alfalfa**

Alfalfa, well known for its extensive ecological adaptability, with development of modern breeding and genetic engineering techniques has advanced in reproductive ecological aspects. Genetic engineering approaches in alfalfa breeding is utilized to manipulate the expression of genes involved in metabolic pathways to improve the reproductive traits, pollination and seed quality traits.

## **3.1 Breeding and seed production**

Any alfalfa cultivar has around 10–200 parents, crossed in isolation to produce breeder seeds. A group of plants distinct for a specific trait, preserved through 2–3 generations from other alfalfa cultivars and cultivars produced by this kind of breeding is called synthetic cultivars. Most commercial alfalfa cultivars are synthetics produced from advanced progenies of clones of superior traits [73]. The out-crossing nature of the alfalfa plant and its polyploid genome create complexity in genetic improvement for higher forage and seed production. The prevalence of severe inbreeding depression prevents researchers from capturing heterosis in alfalfa cultivars through hybrid development. Consequently, it has led to a modification in breeding strategy for higher yield, by intercrossing selected parents to produce synthetic cultivars [74]. This strategy is currently more feasible than the development of a hybrid cultivar. The intercrossing approach between plants with a broad genetic base increase heterozygosity, which increases the intra-locus interaction, and ultimately yield.

### *3.1.1 Alfalfa, a potential plant for genetic research*

The genus *Medicago* is scattered worldwide and comprises 83 species [75]. The cultivated alfalfa for fodder purpose is autotetraploid, cross-pollinated and seed propagated [76]. A rich source of natural variation and genetic resources is held

by three species *M. sativa*, *M. falcata* and *M. media*, with breeding potential for development of new cultivars. Genetic diversity studies of any species are indispensable for successful breeding into new cultivars. Variations of a population, genetically and phenotypically give insights in evolutionary history [77]. A study with 25 accessions of the three *Medicago* species collected in Leh Region, Kashmir, India, analyzed using simple sequence Repeats (ISSRs) and random amplified polymorphic DNA revealed a high genetic variation. A genetic diversity of 30.23% within populations with a mean coefficient of differentiation (Gst) was noticed. The overall value of mean estimated number of gene flow (Nm = 8.0682) revealed large gene exchanges among populations. Analysis of molecular variance (AMOVA) states the genetic diversity being 49% among populations and 51% between populations. The distinct genetic variation in *Medicago* species is an ideal plant for genetic research for breeding for improved forage varieties.

#### *3.1.2 Breeding to overcome inbreeding expression*

Several breeding programs are structured to avoid or overlook the negative impacts of inbreeding expression in alfalfa [72]. Inbreeding expression occurs due to continuous selfing. The vigor of natural tetraploid alfalfa is reduced by inbreeding depression, but maximizing heterozygosity offers a better solution [92]. The intra-locus interaction and additive variation maximizes heterogeneity in alfalfa, to enhance its performance [78]. The forage yield of alfalfa is experienced due to increased frequency of favorable alleles and utilization of non-genetic effects in a study for long years (1898–1985). The study also revealed the enhanced genetic load in recent alfalfa cultivars due to several crossings, which hinders the lethal alleles at their heterozygous loci, providing better germplasm source of alfalfa [79].

#### **3.2 Advances in floral biology**

#### *3.2.1 Effect of anatomical structure of the alfalfa flower in tripping mechanism*

Alfalfa possess a highly unique papilionaceous flower with an exclusive tripping mechanism, which permits only certain insects to enter in to effect tripping [94]. *M. sativa* was studied at the blossom stage to correlate the part of flower tripped by pollinators in relation to the blossom characters. The comprehensive study on the anatomical structure of flowers of 11 selections displayed two chief services involved in tripping mechanisms *viz.*, a cohesive force between the two keel petals and the pressure exerted by the sexual column from cells under tension at the point of fused filaments and the keel. The initial force was resulted due to the interlocking of the projections on the pressed surfaces of keel petals, which is adequate to prevent spontaneous tripping of alfalfa flowers. The firmness of the preventive force and the relative pressure exerted by the sexual column against the appressed keel petals differs in flowers from various selections which is attributed to differences in anatomical structure and development of blossoms. The results revealed that the preventive force of the adhered keel petals is closely related to the proportion of insect tripped flowers than the force with which the sexual column is released by tripping [80].

#### *3.2.2 Mutants of varied inflorescence patterns in alfalfa*

Alfalfa is a typical leguminous plant with a typical raceme inflorescence. The multi-inbreeding process in this cross-pollinated species, develop several mutant forms with long, panicle-like racemes, with fertile and sterile flowers, complex

#### *Reproductive Ecology of Forage Alfalfa (*Medicago sativa *L.): Recent Advances DOI: http://dx.doi.org/10.5772/intechopen.100640*

branched racemes, fascinated racemes etc. The transitional form of some mutants is isolated by means of pair hybridization and new mutant forms were developed. New mutants with diverse inflorescence pattern provide room for more pollinator's activity leading to improved seed set. *Medicago trunculata* shared highly conserved nucleotide sequence and exhibit perfect synchrony between genomes. *M. trunculata* mutant *mtpim* has a complicated inflorescence resembling panicle, controlled by spatiotemporal expression of MtTFL1, MtFULC, MtAP1 and SG1 through reciprocal repression. Some of them resemble *M. sativa* phenotypes. The mutant developed by retrotransposon insertion mutagenesis sg1l-1 has a cauliflower type phenotype resembling a mutant of alfalfa [81]. The data generated on genes regulating inflorescence developed in *Medicago* species helps to understand the phenomenon of inflorescence mutations in alfalfa, which is helpful for modification in inflorescence structure for enhanced pollination.

#### *3.2.3 Ovule sterility and seed set in alfalfa*

The seed potential of alfalfa is very low; an estimated seed to ovule ratio is about 0.08 [82]. This deficit is due to the low number of seeds produced per pod; of 10 ovules present in a floret, only an average of 5 develops into seed. Eliminating at least few causes that limit seed potential will be beneficial in improving seed yield of alfalfa. An ovule sterility trait allied with limiting integument formation controlled by a single recessive gene has been developed in alfalfa. In alfalfa cultivar, Blazer XL, an ovule sterility trait B17, with 81% ovules displaying heavy callose deposition at the time of anthesis with low female fertility was reported [83]. These plants were female sterile when hand crossed with unrelated male fertile plant. In high percentage of ovule sterile plants, under sized pistils develop at anthesis, that will not emerge from staminal column. A mapping study of chromosome region revealed and explains a major share of variation for ovule sterility and the cytological analyses showed that no embryo sac develops in sterile ovules and the callose deposition begins after meiotic division [84], affecting the integumentary tapetum and nuclear cell walls, which usually expands to fill the space occupied by the embryo sac [85]. The pistil growth often ceases at bud stage and a short-aborted pistil is found within the staminal column at anthesis, in a plant with 100% sterile ovules [86]. Nine populations analyzed for ovule sterility showed 4–26% sterile ovules with significant negative correlation between percentage of sterile ovules and seeds per pod in most populations [87]. A quick stain clearing technique based on callose fluorescence is effective to trace ovule sterility in breeding programs. In addition, checking ovule fertility in parental genotypes aid breeders to develop good seed yielding cultivars.

#### *3.2.4 Effect of floral nectaries and flower aroma of alfalfa*

Alfalfa secrets nectar at a uniform daily rate for 4–5 days after flower opening. The volume of the nectar produced differs per floret, which are found to be heritable. Two plants each from a high, intermediate and low nectar producing alfalfa cultivars were subjected to light and scanning electron microscopy, and the images designated nectary located on the staminal column of the receptacle [88]. It comprises of several cell layers subtended by vascular bundles containing both xylem and phloem, but not extend into nectariferous tissue. The epidermal layer comprises permanently open stomata, which functions in nectar secretion. The number of stomata per nectary among the six clones ranged from 24.7 ± 1.9 to 6.8 ± 0.5. The nectar-reservoir diameters ranged from 1.07 ± 0.09 mm to 0.70 ± 0.01 mm. The cultivars with the largest nectar reservoir witnessed high number of stomata [89].

Flower aroma of alfalfa has a specific role in pollinator attraction. Seven clonal lines of alfalfa with difference in flower aroma were consecutively recorded for honey bee visits at different locality and volatiles were also extracted from the flowers. A gas chromatographic study suggests a difference in volatile components [90]. Therefore, the compounds present in the volatiles of nectar has a major role in pollinator attraction for enhanced seed set.

#### *3.2.5 Features of alfalfa flower that effect seed production*

The characters and properties of alfalfa flowers investigated to determine their role in seed setting revealed that the affluence to trip the flower is allied with the age of floret. The age of floret does not have an effect in self-fertilization, but declines for 3 days in cross fertilization. Pollen from untripped florets showed decline in germination, but remained viable when stored in honey bee colony. The honey bees that collect pollen clean themselves with pollen in 2 days. There was no steadfast increase in seed production due to repeated visits of honey bees. Dusting of foreign pollen before tripping has not increased cross fertilization, but 50% increase in self-fertilization. Ovules of florets with exposed stigmas remained functional up to 6 h and then declined rapidly for next 2 days. Self and cross-fertilized seeds were amalgamated in the pods but self-fertilized seeds did not occur beyond the fourth position from the proximal end. Studies suggested that the honey bee pollination can be improved by providing an alfalfa flower with an exposed stigma [91].

#### **3.3 Influence of pollinators in alfalfa pollination and potential for gene flow**

Diverse pollinators have diverse roles in pollinating specific crops. Though managed pollinators, are utilized for pollination of alfalfa for seed production, several wild pollinators also visit them [92–95]. Diverse pollinators display assorted efficiency in depositing and detaching pollen from individual flowers [96, 97]. The rate of tripping and the quantity of pollen deposited varies between pollinator species visiting the racemes [98], which depends on whether the pollinator forages for pollen or for nectar [99] and influence on seed set. Apart from influencing pollination, the pollinators differentially affect gene flow [100]. The bumble bees carry pollen for short distances, but not the hawkmoths. In addition to pollinators, various features of field that grow alfalfa also plays a major role. Increased density of plants has displayed reduced gene flow as pollinators respond to locally available floral resources and shorten the flight distance [101–103]. The load of pollen carried by the pollinators from donor to recipient is expected to turn over quickly as pollinator visits greater number of plants per unit distance traveled, which declines the gene flow.

Though transgenic alfalfa plants are developed, the impact of pollinators on gene flow is yet to be studied as various wild insect pollinators contribute to pollination and movement of genes [104]. A model to predict gene flow between transgenic and conventional field [105], predicted the number of visits by the pollinator in a foraging session, estimates the extent to which the transgenic pollen is diluted by conventional pollen. Essentially, the amount of transgenic pollen on conventional field flower is inversely proportional to the total amount of pollen delivered by each bee during a foraging session in conventional alfalfa. Accordingly, the greater number of flowers the specific pollinator visits, the minimal amount of transmission of transgenic pollen and its fruit set [106]. A study to investigate the involvement of managed honey bees (*A. mellifera*) and leafcutter bees (*Megachile rotundata*), three bumble bee species (*Bombus impatiens*, *Bombus griseocollis*, *Bombus auricomus*) and two solitary bee species to pollination (*Halictus rubicundus* and *Andrena asteris*) and

their contribution in gene flow revealed the potential role of two wild solitary bees and a wild bumble bee in tripping, while the managed pollinators, *A. mellifera* and *M. rotundata* recorded the least. Honey bees, recorded the best potential in gene flow and reduced transgene transmission in relation to other pollinators. The denser plant stand of alfalfa does not show any impact in gene flow and reduced transgene transmission, while the three bumble bee species portrayed an increased gene flow and reduced transgene escape in high density alfalfa plantations [107].

### *3.3.1 Gene flow in commercial seed production*

Alfalfa being an outcrossing and insect pollinated crop, the potential for gene flow has been widely recognized. Gene flow is the exchange of genes from one population to another. It is a natural mechanism that changes the genetic frequency of population over time to enrich the wealth of biological diversity. The formation of cross-pollinated genes and their establishment mediates occurrence of real gene flow [108]. Though pollinator species carry pollen to long distances, true gene flow can occur if the pollen can produce viable seeds and thus offspring. Pollen mediated gene flow is greatly dependent on physical isolation distance between two populations, the degree of synchronous flowering, availability of pollinator gene frequency etc. [109]. Maintaining adequate isolation distances between any alfalfa and seed production trials help to minimize the bee-mediated gene movements. A minimum of 50 m between the alfalfa crops for certified seed production and 200 m for plots exceeding 5 acres and 300 m for plots of 5 acres or less for foundation seed production is recommended [110, 111]. In seed alfalfa production for commercial purpose, bees are purposely stocked in the field. Gene flow, rate of outcrossing, pollinator type and behavior are very important for proper management of commercial alfalfa seed production. Advanced genetic technologies offer better tools to understand the dynamics of pollinator-mediated gene flow. A study to measure the gene flow using honey bee pollination under commercial alfalfa seed production reported a significant decrease in gene flow with increase in isolation distance. A 900 ft. colonies of honey-bee (*A. mellifera*) mediated gene flow was 1.49% and it decreased linearly to 0.20% near 5000 ft. Gene flow continue to decline with increase in isolation distance [112]. The same was reported for alfalfa leaf cutter bee (*M. rotundata*) [113]. Another research with Roundup Ready Trait as a marker gene flow examined the movement of the gene *epsps* from the Roundup Ready plots to conventional alfalfa plots isolated by 152, 274, 610 and 825 m distance. With leaf cutter bee as the pollinator, the pooled data from 2000 to 2002 revealed that the upper bound (99.9% confidence) of gene flow at 274 m was 0.3%, at 152 m was 1.7% and gene flow was not detected at 610 and 825 m at years 2000 and 2002. The results confirmed the role of isolation distance as an effective means to maintain purity of both Roundup Ready and conventional alfalfa plots [114].

#### *3.3.2 Gene flow in forage alfalfa production*

Pollen flow or dispersal is a form where genes can move between plants. However, the total pollen dispersed will not cause gene flow, which is the successful transfer of genetic material. As pollen-mediated gene flow occurs between sexually compatible plants and deposited on stigma of a plant, it fertilizes the ovule, resulting in production of viable seed. Flowering in alfalfa reduces the forage and nutritive value and it has significant consideration in gene flow of alfalfa. Alfalfa managed for forage production is cut several times a year from two to ten, depending upon the climatic factors of the region and the stage of alfalfa development which is early to 10% blooming stage. Periodical harvesting removes the entire plant canopy with flowers or seeds. For high quality forage, first cutting occurs in mid to late bud stage [115]. The cutting interval is 28–35 days, an interval well adequate to initiate full bloom and matured seed. Harvest of alfalfa as dairy feed eliminated the entire plant canopy terminating bloom and seed formation. Regrowth of canopy reinitiated from vegetative crown buds and elongation of lower stem axillary buds. Therefore, alfalfa managed for forage production will have minimal contribution to gene flow, as flower blooming happens in a very less percent and thus pollen transfer. Gene flow from Roundup trait alfalfa to conventional alfalfa for forage production is predicted to be far less as compared to seed production alfalfa [116]. Accordingly, the gene flow from genetic engineered alfalfa grown for forage to conventional forage field is lower than alfalfa grown for seed.

#### **4. Climate change**

Alfalfa, extends its timely service in climate regulation by drawing carbon from atmosphere and dumping deep in soil. But climate change effected by global warming and enhanced nitrogen deposition directly or indirectly affect the reproductive characters of alfalfa. The shift in phenology and foraging crop distribution of plants and pollinators leads to temporal decoupling and spatial mismatch between them [117]. Climate change may affect the floral traits such as floral display, corolla structure, formation and position of ovule and stamen, subsequently affecting the quality and quantity of forage of pollinators, ultimately the reproductive success in plants. Insect pollinators are valuable resources but are limited. Although around 20,000–30,000 bee species are present worldwide, only 10–11 species are managed [118], and *A. mellifera* is far being the most dominant species managed globally. Biotic stress convoyed with climate change causes severe decline in pollinator population. The genus *Apis* is in risk due to climate change and the mismatch in foraging plants and pollinator regimes, thus there is a want for alternate pollinators. Well-known pollinator, *A. mellifera* is being replaced by leaf cutter bee (*M. rotundata*) and alkali bees (*Nomia melanderi*) in alfalfa ecosystem [119].

#### **5. Future scientific challenges and perspectives**

Alfalfa cultivars are usually synthetic populations that originated by a heterogenous blend of heterozygous genotypes, that thwarts the genomic results in breeding process. The application and implementation of genomic techniques for genetically improved alfalfa may be quite promising and challenging, as most matters could be solved at technical and commercial level. Even though, plant genetic engineering encounters the chances for improvement of alfalfa cultivars to some extent, but there are still challenges [120]. Gene transfer techniques involve appropriate use of promoters, transit peptides, choice of selectable or reporter markers etc. The promotors specific to alfalfa are inadequate and fully characterized expressing genes in high level. Consequently, a convention of constitutive, specific promoters effective to alfalfa are mandatory for augmented expression of transgenic research. Therefore, a set of constitutive, tissue or temporal-specific promoters effective in alfalfa is obligatory for the optimized expression of transgenic research [121]. As alfalfa is tetraploid, the transgene integration and gene stacking techniques utilized for other crops are not suitable for alfalfa. Innovative breeding approaches would be needed to adopt and address these challenges in alfalfa.

*Reproductive Ecology of Forage Alfalfa (*Medicago sativa *L.): Recent Advances DOI: http://dx.doi.org/10.5772/intechopen.100640*
