FIELD OF INVENTION:
The present invention relates to the production of a unique Pongamia pinnata plant designated as NANDAN-21 for early flowering (from second year) and high seed yield through mutagenesis via gamma irradiation .

BACKGROUND AND PRIOR ART OF THE INVENTION:
Pongamia  scientifically known as Pongamia pinnata  is an important tree/ shrub of Leguminaceae family. Pongamia is an outbreeding diploid  with a diploid chromosome number of 22. It has wide distribution from India  through central and south-eastern Asia  Indonesia and into northern Australia. Most of the tree is used either in indigenous medicine or for other purposes. The seed’s oil is reddish brown  bitter  thick  non-drying  non-edible and the bitterness is due to flavonoids. The oil has a high content of triglycerides  and its disagreeable taste and odor are due to bitter flavor constituents  pongamin and karanjin. The oil is used for tanning leather  soap making  lamp oil  lubricants  as a liniment to treat scabies  herpes  and rheumatism and also as illuminating oil.

The leaves are known to have antiseptic properties and the presscake leftover from oil extraction is used as a liniment. The roots are used to treat abscesses and as fish poison. The wood is not durable but is used for cabinet and tool-making. It is also an important source of firewood in arid areas (Orwa et al.  2009). The leaves and flowers are high in nitrogen and can be used to enrich soil (Morton  1990). In India  Pongamia is used in land reclamation and as a soil stabiliser. It is being trialled for ‘phytocapping’ landfill waste in Rockhampton (Venkatraman & Ashwath  2009).

It is one of the few nitrogen fixing trees to produce seeds containing 30-40% oil  thus an important and potential new source of biofuel across world. It is known for sustainable rural fuel source  combined with its ability to establish on low fertility soils in India. Selected cultivars of Pongamia are reported to yield seeds up to 40% seed oil. Research and field trials to assess the species’ capacity to produce biofuel are currently underway in Queensland and the Northern Territory. Development of Pongamia as a biofuel crop has largely been centred in India  where scientists have been assessing new crops to support sustainable rural development since the 1990s. The plant has attracted research interest due to its reputation as a drought tolerant crop and its traditional use as a source of oil for lamp fuel (Tigunait  2006; Sarnaik et al.  2010). Preferred habitats include coastal and riverine habitats  primarily in humid tropical and subtropical areas (500–2500 mm rainfall per annum). Pongamia tolerates a wide range of soils  including saline soils. It also produces large numbers of water-dispersed seeds. Its leaves and seeds are toxic to herbivores.

Pongamia is a medium-sized  fast-growing tree or shrub (15–25 m tall). It has been described as briefly deciduous or evergreen with a drooping or spreading branching habit and broad crown (Orwa et al.  2009). The bark is grey or grey-brown and smooth or with faint vertical fissures. The branchlets are hairless with pale stipule scars evident. The leaves are arranged alternately along the stems. Each leaf is composed of 5–9 leaflets with the terminal leaflet being largest. The leaves are hairless  pinkish-red when young  darkening to a glossy green above and a dull green with prominent veins below as they mature. The leaflets are ovate elliptical or oblong (5–25 cm x 2.5–15 cm)  obtuse-acuminate at the apex  rounded to cuneate at the base  not toothed at the edges and slightly thickened. Stipules are caducous.

The inflorescence is raceme-like  6–27 cm long  axillary  pendant and has pairs of strongly fragrant flowers. The flowers are clustered (2–4 together)  short-stalked and pea-shaped (15–18 mm long). The calyx is campanulate  truncate (4–5 mm long) and finely pubescent. The corolla is white to pink  purple inside and brownish-veined on the outside and five-toothed. The lowermost lobe is sometimes longer. The standard is sub-orbicular (1–2 cm)  broad  with basal auricles often with a green blotch and thin silky hairs on the back. The wings are oblong  oblique; with a slightly adherent to obtuse keel (keel petals are coherent at the apex). The stamens are monadelphous; auxillary stamens free at the base but joined with others into a closed tube. The ovary is subsessile to short-stalked and pubescent. There are usually two ovules (rarely three). The style is filiform (with its upper half incurved) and glabrous. The stigma is small and terminal (Duke  1983; Orwa et al.  2010).

Pongamia starts flowering from the 4th or 5th year of planting. Cropping of pods and single almond sized seeds can occur by 4–6 years. White and purplish flowers in axillary recemes appear in April to July. Small clusters of white  purple  and pink flowers blossom on their branches throughout the year  maturing into brown seed pods. Flowering occurs throughout the year in some parts of the world (Wikipedia  2010) but in late spring and summer in Florida and Queensland (Orwa et al.  2010; CILR n.d.).

Pods are smooth  flattened but slightly swollen  oblique-oblong to ellipsoid (3–8 cm x 2–3.5 cm x 1–1.5 cm)  and slightly curved with a short  curved point (beaked). They are 1–3 seeded  brown  thick-walled and leathery to sub-woody  hard and indehiscent; borne on short stalks in quantities. Seeds are elliptical or compressed ovoid  bean-like  with a brittle coat (1.5–2.5 cm x 1.2–2 cm x 0.8 cm)  flattened  dark brown and oily. Seeds tolerate salt concentrations equivalent to sea water (Royal Botanic Gardens Kew  2002). It has an extensive lateral root system and a long taproot. Pongamia does not appear to require specific inoculation with Rhizobium bacteria (Daniel  1997; Orwa et al.  2009).

Growth is rapid  with specimens reaching adult height in 4–5 years and producing seeds in 4–7 years. Seed production is prolific  with a single tree yielding 9–90 kg of seeds per annum  indicating a yield potential of 900–9000 kg seeds/ha assuming a plant density of 100 trees/ha (Duke  1983). Gresshoff (Rural Diversity  2010) suggests individual trees are capable of producing 30 000 seeds per annum in Australia. In India  research is underway to develop high-seeding varieties with an emphasis on selecting varieties that will yield maximum quantities of biofuel (oil extracted from the seeds) (Sahoo et al.  2009; Mukta Sreevalli  2010; Sunil et al.  2010). One early rural development project—the ‘Seeds of Hope’ project in southern India—found that wild types are unpredictable in cropping (number of pods per tree) and trees can take up to 10 years to mature. The practice of grafting helped reduce time to maturity to three years in some cases (Tigunait  2006). However  economic yields stabilization is from third years onwards.

Breeding objectives depend on use of the specific crop; increasing yield is a primary objective in all programs. Seed yield of Pongamia is determined by number of pods per plant  number of fertile flowers per inflorescence  seed yield per tree and 100-seed weight. As the maximum number of seeds per pod is limited and the agronomic factor of planting density does not offer much flexibility for increasing yields  selection should focus on the other yield components to obtain higher yield. Heritable variation exists for all of these components except number of seeds per pod and breeders may directly or indirectly select for increase in any of them. The goal of Pongamia breeding is to develop new Pongamia tree with early flowering combined with higher yield. The breeder initially selects and crosses two or more parental lines  followed by selection among the many new genetic combinations. The breeder can theoretically generate billions of new and different genetic combinations via crossing. The breeder has no direct control at the cellular level; therefore  two breeders will never develop the same line  or even very similar line  having the same traits of Pongamia.

Choice of breeding methods to select for the improved combination of traits depends on the mode of plant reproduction  the heritability of the traits being improved  and the type of cultivar used commercially. For highly heritable traits  a choice of superior individual plants evaluated at a single location will be effective  whereas for traits with low heritability  selection should be based on mean values obtained from replicated evaluations of families of related plants.

Mutation induction has become a proven way of creating variation within a crop variety. It offers the possibility of inducing desired attributes that either cannot be found in nature or have been lost during evolution. When no gene  or genes  for resistance to a particular disease  or for tolerance to stress  can be found in the available gene pool  plant breeders have no obvious alternative but to attempt mutation induction. Treatment with mutagens alters genes or breaks chromosomes. Gene mutations occur naturally as errors in deoxyribonucleic acid (DNA) replication. Most of these errors are repaired  but some may pass the next cell division to become established in the plant offspring as spontaneous mutations. Gene mutations without phenotypic (visible) expressions are usually not recognized. Consequently  genetic variation appears rather limited  and scientists have to resort to mutation induction. The mutation techniques have significantly contributed to plant improvement worldwide  and have made an outstanding impact on the productivity and economic value of some crops (Ahloowalia et al.  2004).

Mutation is any heritable alteration in genetic material includes such diverse phenomenon as change in the number of chromosomes  changes in the structure of chromosomes and changes within the genes themselves. Although change in the number and structure of the chromosomes are of considerable importance to the evolutionary geneticist and to the plant breeder. Mutation always occurs within a gene and it should be the smallest possible change in the structure of the genetic material that is detectable as a mutation. Every gene has its own characteristic rate of mutation  some genes mutating more frequently than others. Gamma irradiations were also found to cause modulation in protein patterns by inducing appearance and/or disappearance of some protein bands  concluded by Rashed et al. (1994). Yoko et al. (1996) studied the effect of gamma irradiation on the genomic DNA of corn  soybeans and wheat. They concluded that  large DNA strands were broken into small strands at low irradiation dose but small and large DNA strands were broken at higher irradiation doses. The RAPD method was also used by Raisheed et al. (2001) to detect the genetic variation induced by gamma rays. Radiation by gamma rays leads to increasing the level of DNA break formation. These different types of DNA damages could be detected by changes in RAPD profiles (Senthamizh et al.  2008).

Pongamia is an important and valuable oil seed crop. The natural constraints that limit its large-scale production and availability  to meet the demand for biodiesel production  are its long gestation period (4–7 years)  plant height  seed storage behaviour  insect pests  and the seed oil yield and quality (NOVOD  2010). The cross-pollinating nature of M. pinnata contributes to its wide germplasm biodiversity. Thus  it becomes an important step to examine the genetic variations among naturally growing elite individuals of M. pinnata at inter- and intra-population levels  and to prepare strategies for its specific exploitation by plant breeders in promoting it as a versatile biodiesel plant. Thus  a continuing goal of Pongamia breeders is to develop early flowering and high yielding plants that are agronomically potential. The reasons for this goal are to maximize the amount of seed oil produced in order to provide an alternative source for replacing the deficit in the use of fossil fuel and to boost the supply of biofuels. To accomplish this goal  the Pongamia breeder must select and develop Pongamia plants that have the traits to result in early flowering and superior yields.

The following are the patents either issued or in the process of issuing related to Pongamia  but none of them are citing about plant material improvement for early flowering and higher seed yield.

Indian Patent Application No. 1084/CHE/2009 discloses a process for pretreatment of Jatropha/Pongamia seed before oil expulsion to reduce free fatty acids in oil and to increase oil yield for higher Biodiesel recovery applied in May 2009.

Energy Efficient Mini oil expeller for extraction of Pongamia and Jatropha oils at CRIDA  Rajendranagar  Hyderabad  India.

Mini oil expeller: A mini oil expeller with low energy input for higher oil recovery for Pongamia and Jatropha oil extraction is designed and developed at CRIDA  Rajendranagar  Hyderabad  India.

Indian Patent Application No.1390/CHE/2005 A discloses anti-viral agent from the plant Pongamia pinnata and a process for the preparation of the said agent.

Investigations on emission characteristics of the Pongamia biodiesel–diesel blend fuelled twin cylinder compression ignition direct injection engine using exhaust gas recirculation methodology and dimethyl carbonate as additive by M. Pandian et al.  2010 is published in J. Renewable Sustainable Energy 2  043110 (2010); doi:10.1063/1.3480016 (12 pages).

Trial plantations at Katherine in the Northern Territory were found to flower profusely but did not set large numbers of pods. In contrast  Queensland varieties were found to produce fewer flowers but more pods. Highest yielding individuals are reported from Queensland and these superior lines have now been introduced to the Northern Territory for trials (Bennett  2009). Clonal material is being produced in Queensland (Walkamin) for bulk sale and individual lines have been selected for superior early flowering  crop canopy  yield and oil content (Clonal Solutions  2010). Vegetative propagation can occur from cuttings and root suckers (new plants growing from lateral roots of the parent tree). At times  root suckers may be prolific (Orwa et al.  2009).

No patent prior art or publication is available on development of early flowering and high yielding Pongamia pinnata plant. Hence  the invention has great significance for early flowering (from second year)  increased yields and consistent feed stock supply for biodiesel production. The NANDAN-21 has socio-economic and environmental benefits globally  for supporting developing economies by utilizing marginal lands for energy production and supply.

OBJECT OF THE INVENTION:
The object of invention is to develop an early flowering (shortest period) and high seed yielding Pongamia pinnata plant  NANDAN-21 through gamma irradiation mutagenesis of selected accession of Pongamia pinnata from NANDAN farm and molecular confirmation of NANDAN-21.

SUMMARY OF THE INVENTION:
In accordance with the above objectives  the present invention discloses a Pongamia plant designated as NANDAN-21  responsible for early flowering (from second year) with high yield and molecular characterization of NANDAN-21.

The following embodiments and aspects thereof are described in conjunction with systems  tools and methods which are meant to be exemplary and illustrative  not limiting in scope. In various embodiments  one or more of the above described problems have been reduced or eliminated  while other embodiments are directed to other improvements.
The present invention relates to a Pongamia plant designated as NANDAN-21 and plants (clones) derived from NANDAN-21. The invention also relates to the selected plants and any further progeny or descendants of the hybrid derived by crossing NANDAN-21 as a pollen donor. The invention is also directed to methods for producing a Pongamia plant by irradiating with gamma rays followed by forwarding till three generations with periodical observations. Thus  any methods using the selected Pongamia plant NANDAN-21 in backcross  hybrid production  crosses to population  clonal propagation  micro propagation and the like are part of this invention. All plants which are a progeny of or descend from NANDAN-21 are within the scope of this invention. It is an aspect of this invention for NANDAN-21 to be used in crosses with other  different Pongamia plants to produce first generation (F1) Pongamia hybrid seeds and plants with superior characteristics.

In another aspect  the present invention provides regenerable cells for use in tissue culture (micro propagation) of NANDAN-21. The tissue culture will preferably be capable of regenerating plants having the physiological and morphological characteristics of the foregoing Pongamia plant  and of regenerating plants having substantially the same genotype as the foregoing Pongamia plant. Preferably  the regeneration cells in such tissue cultures will be embryos  protoplasts  meristematic cells  callus  pollen  leaves  anthers  gynoecium  root tips  seeds or stems. Still further  the present invention provides selected Pongamia  NANDAN-21 plants regenerated from the tissue cultures of the invention.

In addition to the exemplary aspects and embodiments described above  further aspects and embodiments will become apparent by study of the following descriptions.

BRIEF DESCRIPTION OF FIGURES:
FIGURE 1: Is a file photo of the NANDAN-21 Pongamia mutant showing exhaustive flowering in a two year Pongamia pinnata plant.

FIGURE 2: PCR amplification of Pongamia NANDAN-21 samples and control samples using RAPD-OPQ16 primer specific to the Pongamia NANDAN-21 mutant.

Lane description: 1 – 1 kb DNA ladder (Fermentas); 2 – Empty lane; 3 – Pongamia (Control); 4 – Pongamia (Control); 5 – Pongamia mutant (150 Gy); 6 – Pongamia mutant (150 Gy); 7 – Pongamia mutant (150 Gy); 8 – Empty lane; 9 – 1 kb DNA ladder (Fermentas)

FIGURE 3: Sequence listing of Nandan- 21 (441 bp)

DETAILED DESCRIPTION OF THE INVENTION:
DEFINITIONS
In the description and tables which follow  a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims  including the scope to be given to such terms  the following definitions are provided:
Essentially all the physiological and morphological characteristics: - A plant having essentially all the physiological and morphological characteristics mean a plant having the physiological and morphological characteristics of the cultivar  except for the characteristics derived from the hybridization and selection.
• 100-Seed weight: The weight of 100 Pongamia seeds as measured in grams.
• Regeneration: Regeneration refers to the development of a plant from tissue culture.
Pongamia plant  NANDAN-21 is an early flower initiating (from second year) and high yielding plant with more numbers of inflorescences per plant  more number of pods per inflorescence and higher 100-seed weight.

All publications cited herein and incorporated herein by reference for the purpose of describing and disclosing compositions and methodologies that might be used in connection with the invention.

As used herein  the term ‘early flowering’ is used to describe the first initiation of flowering in a well established plant that can be referred to as early flower initiation.

As used herein  the terms “mutated”  “stable mutant” or “mutant” with reference to a plant cell means the plant cell has modified nucleic acid sequence in its genome which is maintained through two or more generations.

As used herein  the term “expression” refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation.

As used herein  the terms “modified” regarding a plant trait  refers to a change in the phenotype of a mutant plant relative to a non-mutated plant  as it is found in nature.

As used herein  the term “M1  M2 and M3 population” refers to generation of mutated plants from the seed of mutant plant. The M1 generation is the first set of mutated plants that can be selected by application of a selection  e. g. a Physiological and morphological characters  oil content analysis  for which the mutant plant contains the corresponding early flowering gene.

As used herein  the term “phenotype” may be used interchangeably with the term “trait”. The term refers to a plant characteristic that is readily observable or measurable and results from the interaction of the genetic make-up of the plant with the environment in which it develops. Such a phenotype includes chemical changes in the make-up resulting from enhanced gene expression which may or may not result in morphological changes in the plant  but which are measurable using analytical techniques known to those of skill in the art.

The present invention describes the methods and material of induced mutation through gamma irradiation of selected accession of Pongamia pinnata from NANDAN farm. The subject invention relates in part to the discovery of an induced mutant of Pongamia pinnata designated as NANDAN-21. The subject invention involves “early flowering” mutation and related inbred development. The subject invention further provides over-expression or up-regulation of dominant gene that responsible for flowering in Pongamia pinnata inbred lines and near isogenic lines. The subject invention also provides a new and distinctive Pongamia pinnata inbred line designated as NANDAN-21. The mutation was discovered in plant no 3-9

NANDAN-21 is originated by gamma irradiation of selected accession of Pongamia pinnata from NANDAN farm breeding population. It was expressing initial flowering period of 5-7 years as the control Pongamia pinnata (Nandan selection). Inheritance of the subject trait conferred by the mutated gene appears to be over-expression or up-regulation of flowering trait responsible gene via gamma irradiation. The effect is seen as over-expressing or dominant expression via early flowering trait  but there are some indications of “gene dosage” effects.

Insertion of this over-expressed gene or modified gene will (by backcrossing  for example) allow the direct use of early flowering Pongamia inbred line in regular flowering Pongamia inbred. The trait responsible gene can also be used for transgenic research and development in other late flower initiating crops. Explicitly  this trait can also be bred or otherwise introduced into other  late flower initiating Pongamia plants.

Further Embodiments of the Invention:
Methods for Mutagenesis
The dry and dormant seeds of selected accession of Pongamia pinnata from NANDAN farm were used for inducing mutagenic treatments through gamma irradiation. Hundred seeds were taken for each treatment. The seeds were packed in polythene bags and were exposed to 100  150  200 and 250 Gy doses of gamma rays in 60Co gamma source. The gamma radiation was carried out at ANGRAU  Hyderabad  India. The irradiated seeds were sown in the field along with the control (non-irradiated seeds) in a randomized block design (RBD) with three replications. All the treatments including control were raised adopting a spacing of 4 mt in between rows and 4 mt in between plants  respectively.
Similar kind of treatment using Ocimum sanctum Linn. seeds  the plants in the mutagen treated populations commenced flowering about 15-20 days earlier than control pants and were categorized as ""Early flowering"" (Nasare and Choudhary  2011). In the control plants  flowering commenced after 40-50 days of germination  whereas in the early flowering mutants started from 19th to 29th day after germination. In some of the early flowering ocimum mutants  the number of branches and the weight of seeds were found to be more than control. The frequency of these mutants ranges from 0.28 to 0.27% in M2 and M3 0.38 to 0.26 in M3 in gamma ray treated population.

The selected accession of NANDAN farm (possessing long flower initation time and normal seed yield) Pongamia seeds subjected to mutagenesis as described above were sown to produce first generation (M1) mature seed-bearing plants. These M1 generation plants produced seeds were used for sowing of second-generation (M2). The M2 generation seeds were harvested and sown to produce seed-bearing M3 plants that produced a third generation (M3) seeds. Each M3 line traced to a different M2 individual plant. Data on the flowering time  number of inflorescences per plant  pods per inflorescence and seed yield was recorded from the M3 generation Pongamia plants.

This invention is directed to methods for producing a NANDAN-21 by gamma irradiation induced mutation to selected accession of Pongamia pinnata plant from NANDAN farm. Therefore  any methods using the mutant NANDAN-21 are part of this invention: selfing  M1 to M3 populations. Any plants produced using Pongamia pinnata hybrid NANDAN-21 as a parent is within the scope of this invention. The inventors have characterized the marker gene  which is supposed to enhance or up-regulate the initial flowering time in Pongamia pinnata plant.

Molecular markers
“Molecular markers” are a tool to study the diversity on the genetic level. For molecular cloning and PCR techniques  DNA markers are a popular means for identification and authentication of plant and animal species. DNA based markers are less affected by age  physiological conditions of samples and environmental factors. They are not tissue specific and thus can be detected at any phase of organism development. Only a small amount of sample does not restrict detection. The power of discrimination of DNA-based markers is so high that very closely related varieties can be differentiated.

Significance of molecular markers
DNA-based molecular markers have acted as versatile tools and have found their own position in various fields like taxonomy  physiology  embryology  genetic engineering  etc. They are no longer looked upon as simple DNA fingerprinting markers in variability studies or as mere forensic tools. Ever since their development  they are constantly being modified to enhance their utility and to bring about automation in the process of genome analysis. The discovery of PCR (polymerase chain reaction) was a landmark in this effort and proved to be a unique process that brought about a new class of DNA profiling markers. This facilitated the development of marker-based gene tags  map-based cloning of agronomically important genes  variability studies  phylogenetic analysis  synteny mapping  marker-assisted selection of desirable genotypes  etc. Thus  giving new dimensions to concerted efforts of breeding and marker-aided selection that can reduce the time span of developing new and better varieties and will make the dream of super varieties come true. These DNA markers offer several advantages over traditional phenotypic markers  as they provide data that can be analyzed objectively. In this article  DNA markers developed during the last two decades of molecular biology research and utilized for various applications in the area of plant genome analysis are reviewed. DNA markers that can be routinely employed in various aspects of plant genome analysis such as taxonomy  phylogeny  ecology  genetics and plant breeding.

Genetic polymorphic markers employ DNA amplification using short primers of arbitrary sequence. These primers have been termed ‘random amplified polymorphic DNA’ or “RAPD” primers  “RAPD amplification” refers to a method of single primer directed amplification of nucleic acids using short primers of arbitrary sequence to amplify nontargetd  random segments of nucleic acid. Williams et al.  Nucl. Acids. Res.  18  6531(1990) and U.S Pat. No. 5 126 239; (also EP 0 543 484 A2  WO 92/03567). The RAPD method amplifies either double or single stranded nontargetd  arbitrary DNA sequences using standard amplification buffers  dATP  dCTP  dGTP and TTP and a thermostable DNA polymerase such as Taq. The nucleotide sequence of the primers is typically about 9 to 13 bases in length  between 50 and 80% G+C in composition and contains no palindromic sequences. RAPD detection of genetic polymorphisms represents an advance over RFLP in that it is less time consuming  more informative  and readily susceptible to automation. Because of its sensitivity for the detection of polymorphisms RAPD/PCR methods have become the methods of choice for analyzing genetic variation within species or closely related genera  both in the animal and plant kingdoms (U.S Pat. No. 5  660  981). “RAPD Primers” refers to primers of about 8 to 13 bp  of arbitrary sequence  useful in the RAPD amplification or RAPD analysis according to the instant method. The “RAPD marker profile” refers to the pattern  or fingerprint  of amplified DNA fragments which are amplified during the RAPD method and separated and visualized by gel electrophoresis.

Randomly-Amplified Polymorphic DNA Markers (RAPD). In 1991  Welsh and McClelland developed a new PCR-based genetic assay namely randomly amplified polymorphic DNA (RAPD). This procedure detects nucleotide sequence polymorphisms in DNA by using a single primer of arbitrary nucleotide sequence. In this reaction  a single species of primer anneals to the genomic DNA at two different sites on complementary strands of DNA template. If these priming sites are within an amplifiable range of each other  a discrete DNA product is formed through thermo cyclic amplification. On an average  each primer directs amplification of several discrete loci in the genome  making the assay useful for efficient screening of nucleotide sequence polymorphism between individuals. However  due to the stochastic nature of DNA amplification with random sequence primers  it is important to optimize and maintain consistent reaction conditions for reproducible DNA amplification. They are dominant markers and hence have limitations in their use as markers for mapping  which can be overcome to some extent by selecting those markers that are linked in coupling. RAPD assay has been used by several groups as efficient tools for identification of markers linked to agronomically important traits  which are introgressed during the development of near isogenic lines. The application of RAPDs and their related modified markers in variability analysis and individual-specific genotyping has largely been carried out  but is less popular due to problems such as poor reproducibility faint or fuzzy products  and difficulty in scoring bands  which lead to inappropriate inferences.

The term “Primer” refers to a nucleic acid fragment or sequence that is complementary to at least one section along a strand of the sample nucleic acid  wherein the purpose of the primer is to sponsor and direct nucleic acid along that string. Primers can be designed to be complementary to specific segments of a targeted sequence. In PCR  for example  each primer is used in combination with another primer forming a “Primer Set” or “Primer Pair”  this pair flanks the targeted sequence to be amplified. In RAPD amplification  single arbitrary primers are used to amplify non targeted segments of nucleic acid which are located between the primer sequence sites in opposing DNA strands. The term “primer” as such  is used generally herein by Applicants to encompass any sequence-binding oligonucleotide which functions to initiate the nucleic acid replication process. “Diagnostic primers” will refer to primers designed with sequences complementary to primer binding sites on diagnostic marker. Diagnostic primers are useful in the convenient detection and identification of individuals of a genetically related population.

PCR-based markers
PCR Enzymatically multiplies a defined region of the template DNA (Fig. 1). The specific multiplication is attributed to the presence of primers  which are single-stranded polynucleotide that recognize and bind to the complementary DNA sequence on template DNA. The amplification process starts with denaturation of the double-stranded template DNA to single stranded DNA under a high temperature  usually between 90–95°C  followed by the specific annealing of the primer(s) to the single-stranded template DNA at a lower temperature. The annealing primers are then extended by a thermo stable DNA polymerase. Repeating the denaturation-annealing extension cycle leads to an exponential accumulation of the DNA fragment of the defined sequence. The amplified products are then fractionated on agarose  polyacrylamide or other gel matrix and detected by Ethidium bromide (EtBr) or silver staining  autoradiography (using an isotope-labeled primer)  or fluorescence (using a fluorescence-labeled primer). The distance between the priming sites is usually from 100 bp to a few kilo bases (kb) (and hence the size of the amplified fragments)  although the recently developed ‘long distance PCR’ allows amplification of up to 40 kb or beyond. PCR amplification of any region of a DNA sample is possible  providing their flanking sequences are known  as these are needed for designing primers. Owing to PCR’s sensitivity and ability to amplify DNA from a small amount of materials in vitro  a variety of PCR-derived methods have been established.

Hybridization-based marker technologies use cDNA  cloned DNA elements  or synthetic oligonucleotides as probes  which are labelled with radioisotopes or with conjugated enzymes that catalyze a coloured reaction  to hybridize DNA. The DNA is cleaved with restriction enzymes or amplified by PCR  separated by gel electrophoresis  and transferred to a solid support matrix.

Identification of the Marker:
Molecular Characterization of NANDAN-21
Plant Material
Attempts were made to develop diagnostic molecular markers for the early flowering and high seed yielding selected plant  NANDAN-21. For this purpose  molecular analysis of the plant NANDAN-21 in comparison with Milettia pinnata normal plant (exhibiting initial flowering period of 5-7 years  number of inflorescence per plant  number of pods per inflorescence and seed yield per pant).

DNA Extraction
Total genomic DNA was extracted from younger leaves of the selected plant (NANDAN-21) from M3 generation irradiated Pongamia plant and control Pongamia non-irradiated plant following the standard CTAB method with minor modifications (Doyle and Doyle  1987). Five grams of leaves were ground in liquid nitrogen  then homogenized in 20 ml of extraction buffer (2% CTAB  20 mM EDTA  2% PVP  1.4 M NaCl  100 mM Tris-HCl pH 8.0 and 1% ?-mercaptoethanol) and incubated at 65 ºC for 1h. The supernatant was treated with RNase A (100 ?g/ml)  incubated at 37 ºC for 30 min and twice extracted with chloroform: isoamylalcohol (24:1 v/v). The DNA was precipitated with isopropanol and washed twice with 70% ethanol. The pelleted DNA was air dried and resuspended in 500 ?l of sterile Millipore water and stored overnight at -20 ºC.

RAPD and PCR Analysis
A total of 200 decamer primers from Operon kits - OPB to OPK (Operon technologies  Alameda  USA) were used for DNA amplification according to the method of Williams et al. (1990). The PCR amplification reaction (10 ?l) consisted of 2.5 ng of DNA  1x PCR buffer (10 mM Tris pH 9.0  50 mM KCl  1.5 mM MgCl2)  100 ?M of each of the four dNTPs  0.4 ?M of RAPD primer and 0.3 U of Taq DNA polymerase (Bangalore Genei  India). PCR amplifications were performed in an Gene Amp 9700 Thermal Cycler (Eppendorf) with an initial denaturation at 94 ºC for 3 min followed by 45 cycles at 94 ºC for 45 s  36 ºC for 30 s and 72 ºC for 2 min with a final extension at 72 ºC for 7 min. The PCR products were separated on 1.5% agarose gel in 1x TAE buffer by electrophoresis at 100 V for 3 h and visualized with ethidium bromide staining under gel documentation system. In general  RAPD markers suffer from a lack of reproducibility  but to check the consistency of the electrophoretic patterns and the polymorphism detected  every PCR reaction was repeated twice. All the PCR amplifications included a negative control (no DNA) to avoid erroneous interpretations.

The 200 tested primers gave robust amplification profiles. The polymorphism detected and Polymorphic bands were checked for specific bands. Only one marker was found specific to NANDAN-21.

Further  the distinction of NANDAN-21 has been accomplished through development of a molecular marker specific to the hybrid. The molecular marker gives a specific band of 441 bp (Primer) in NANDAN-21 (Figure 2 and Figure 3). Results indicated the association of the marker with the plant of interest.

TISSUE CULTURE
As used herein  the term ‘tissue culture’ indicates a composition comprising isolated cells of the same or a different type or a collection of such cells organized into parts of a plant. Exemplary types of tissue cultures are protoplasts  calli  plant meristems  and plant cells that can generate tissue culture that are intact in plants or parts of plants  such as embryos  pollen flowers seeds  racemes  leaves  stems  roots  root tips  anthers  and the like. Means for preparing and maintaining plant tissue culture are well known in the art. By way of example  a tissue culture comprising organs has been used to produce regenerated plants.
As used herein  the term ‘plant’ includes plant cells  plant protoplasts  plant cells of tissue culture from which Pongamia plants can be regenerated  plant calli  plant meristems  and plant cells that are intact in plants or parts of plants  such as pollen  flowers  embryos  ovules  seeds  spike  leaves  stems  gynoceium  anthers and the like. Thus another aspect of this invention is to provide for cells which upon growth and differentiation produce a cultivar having essentially all of the physiological and morphological characteristics of NANDAN-21.

The present invention contemplates a Pongamia plant regenerated from tissue culture of the selected Pongamia plant NANDAN-21 of the present invention. As is well known in the art  tissue culture of Pongamia can be used for the in vitro regeneration of a Pongamia plant. Tissue culture of various tissues of Pongamia and regeneration of plants there from is well known and widely published. For example  reference may be had to Vigya Kesari et al. (2010) Effect of genotype and auxin treatments on rooting response in stem cuttings of CPTs of Pongamia pinnata  a potential biodiesel legume crop. CURRENT SCIENCE  VOL. 98  NO. 9  10 MAY 2010; Kesari  V.  Krishnamachari  A. and Rangan  L.  Effect of auxins on adventitious rooting from stem cuttings of candidate plus tree Pongamia pinnata (L.)  a potential biodiesel plant. Trees – Struc. Func.  2009  23  597–604 and Mesen  F.  Leakey  R. R. B. and Newton  A. C.  The influence of stock plant environment on morphology  physiology and rooting of leafy stem cuttings of Albizia guachapele. New Forests  2001  22  213–227. Thus  another aspect of this invention is to provide cells which upon growth and differentiation produce Pongamia plants having the physiological and morphological characteristics of NANDAN-21.

Additional Methods
Apart from the mutation  tissue culture and regular cultivation methods the Pongamia plant can be propagated through grafting  cuttings and cross breeding with early flowering type Pongamia with high yielding type Pongamia plants. Grafting technique has proved the importance in shortening the initial flowering time  but has several disadvantages like laborious work  skill oriented  needs selection of variants  identification of rootstock  and demands the best sources of high-quality grafting stock. The results obtained out these additional will be not so fruitful because of the skill involved in the techniques  selection of suitable and proficient parent material  success rate of the technique  yield variation from the technique involved  time taken for the technique  economic yield obtained from the process and thereof. So  the additional methods followed will be only an effort to try  but will not come out with any efficient results.
Duke  JA.1983 Handbookofenergycrops unpublished <http://www.hort.purdue.edu/newcr op/duke_energy/Pongamia_pinnata.html>.

Orwa  C  Mutua  A  Kindt  R  Jamnadass  R  Simons  A (2009). Agroforestree Database: a tree reference and selection guide. Version 4.0. http://www.worldagroforestry.org/ treedb2/AFTPDFS/Pongamia_pinnata.pdf) (accessed 8 and 26 March 2010).
Morton  JF 1990  ‘The pongam tree  unfit for Florida landscaping  has multiple practical uses in under-developed lands’  Proceedings of the Florida State Horticultural Society 103: 338–43.
Venkatraman  K and Ashwath  N 2009  ‘Phytocapping – importance of tree selection and soil thickness’  Water  Air and Soil Pollution: Focus 9(5–6): 421–30.
Tigunait  I 2006  ‘Seeds of hope: drought  biofuel and community renewal’  Yoga+ Magazine  Dec–Jan  pp. 78–87.
Sarnaik  J  Godbole  A and Punde  S 2010  Integrating high conservation value native species into biofuel production for conservation and sustainable use of biodiversity  Applied Environmental Research Foundation (AERF)  India.
Nasare P. N. and A. D. Choudhary  2011. Early Flowering and High Yielding Mutants In Ocimum sanctum Linn. Indian Streams Research Journal. Vol. 1  Issue III / April 2011  pp.202-204.

We Claim 

1. The mutant Pongamia pinnata plant designated as NANDAN-21 is produced by induced mutation through gamma irradiation (dose 150 Gy) at M3 generation following selection and self pollination.

2. A Pongamia pinnata mutant plant NANDAN-21 which is responsible for early flowering and high seed yield.

3. NANDAN-21 produced by asexual propagation or tissue culture process or growing the seed of the claim 2.

4. Pollen of the mutant plant (NANDAN-21) of claim 3.

5. An ovule of the mutant plant (NANDAN-21) of claim 4.

6. A complete methods and material of induced mutation through gamma irradiation in Pongamia pinnata to development of NANDAN-21.

7. A Pongamia pinnata (NANDAN-21) plant or a part thereof; is having all of the physiological and morphological characteristics of the NANDAN-21 plant of claim 2.

8. A tissue culture cells produced from the Pongamia pinnata NANDAN-21 plant of claim 2  wherein said cells of the tissue culture are produced from a plant part selected from the group consisting of leaves  pollen  embryos  cotyledons  hypocotyls  meristematic cells  roots  root tips  pistils  anthers  flowers  inflorescence  and stems.

9. A protoplast produced from the mutated hybrid NANDAN-21 plant of claim 2.

10. A marker gene responsible for early flowering in Pongamia pinnata.

11. A mutant hybrid NANDAN-21 plant or part thereof prepared by the process of claim 8 & 9.

12. A method of producing a mutant NANDAN-21 of Pongamia pinnata plant with the morphological and physiological characteristics of according to claim 9 or claim 10  comprising regeneration of a tissue culture.

13. A method of producing a early flowering plant of Pongamia pinnata designated as NANDAN-21 according to any one of claims 9 to 11 wherein the NANDAN-21 exhibits a early flowering and higher seed yield.

14. A method of producing a mutant hybrid of Pongamia pinnata plant designated as NANDAN-21 according to any one of claims 9 to 12  wherein the M1 to M3 population used in the method comprises the early flowering and high seed yield.

15. A early flowering Pongamia pinnata plant comprising a mutated  suppress to single marker gene that confers for early flowering in Pongamia pinnata wherein a major locus for said marker gene can be mapped to one end of linkage group.

16. The early flowering and high seed yielding Pongamia plant of claim 15 over expressing an early flowering gene.

17. A seed produced by the plant of claim 15  wherein said seed comprises the said gene.

18. A progeny plant of the plant of claim 1  wherein said progeny plant comprises the said gene.

19. A method of determining if a genomic test sample comprises a gene capable of conferring a early flowering plant and high seed yield phenotype on a plant  said genomic test sample being obtained from a test plant or tissue  seed  or a part of said test plant  said test plant comprising a genome  and said method comprising assaying said test sample for analysis of Pongamia provides insight into the regulation early flowering  and ultimately to upregulation the early flowering of Pongamia plant.

20. A part of a plant of claim 15  said part comprising said gene.

21. The plant part of claim 19  wherein said part is a seed or pollen.

22. A method of culturing tissue cells from a mutated hybrid of Pongamia pinnata plant according to any one of claims 9 to 18  comprising culturing tissue cells produced from the Pongamia pinnata plant  where cells are selected from the group consisting of leaves  pollen  embryos  cotyledons  hypocotyls  meristematic cells  roots  root tips  pistils  anthers  flowers  inflorescence  callus  seeds and stems.

23. Use of the mutant hybrid NANDAN-21 plant or part thereof  according to claim 22 in a process for the production of a biofuel.

24. Use according to claim 22 wherein the biofuel is biodiesel.

25. A biofuel generated by the use according to claim 27 or claim 28.

Dated this the 7th day of September  2011

Dr. P. Aruna Sree
(Regn.No.: IN/PA 998)
Agent for the Applicant