THE GAZETTE OF INDIA: EXTRAORDINARY (Part II- Sec. 3(ii))

FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
PROVISIONAL/COMPLETE SPECIFICATION
(See section 10 and rule 13)
1.TITLE OF THE INVENTION Method of making resistant genotype for
phomopsis blight disease in brinjal-
eggplant {Solarium melongena L.)
2) APPLICANT (S)
Name: Ankur Seeds Private Limited
Nationality: Indian
Address: Ankur Seeds Pvt. Ltd.
27, New Cotton Market Lay Out
Nagpur- 440018
(MS) India.
3) PREAMBLE TO THE DESCRIPTION

PROVISIONAL
The following specification describes the invention.

COMPLETE
The following specification particularly describes the invention and the manner in which it is to be performed.

4) DESCRIPTION (Description shall start from nest page.)
5) CLAIMS (not applicable for provisional specification. Claims should start with the preamble-' I /We claim ' on separate page)
6) DATE AND SIGNATURE (to be given at the end of last pages of specifications)
7) ABSTRACT OF THE INVENTION (to be given along with compete specification on separate page)

Method of making resistant genotype for phomopsis blight disease in brinjal-eggplaht {Solatium melongena L.)
Field of invention:
This invention relates to the field of plant breeding, disease resistance and plant resistant gene introgression methods. More specifically, an improved method of generating introgressed disease resistant brinjal-eggplant is developed. This has provided an extremely valuable invention from view point of agriculture for breaking the incompatibility barriers, making fertile, self and cross compatible, resistant introgressed cultivars, by virtue of substitute to reduce pesticide use.
Background of invention or prior art:
Brinjal-eggplant {Solanum melongena L.) is an important crop species in Asia, Europe and North America. Brinjal-eggplant has been cultivated in the country for last 4000 years; although it is often thought of as a Mediterranean or Mid- Eastern vegetable. India has been widely considered as a source of origin for brinjal-eggplant. Amongst the solanaceous vegetables, brinjal-eggplant {Solanum melongena L.) is the most common, popular and principal vegetable crops grown in many geographical parts in India. The area under brinjal-eggplant cultivation is estimated at 0.51 million hectare with total production of 8,200,000 Mt. (FAO data, 2004, http://faostat.fao.org./). Brinjal-eggplant is grown by small farmers and is an important source of income. It is a versatile crop adapted to different agro climatic regions and can be grown throughout the year in India. A number of cultivars are grown in the country, as consumer preference being dependent upon the yield of the cultivars, fruit colour, size and shape. It is highly productive crop, the fruits are consumed as cooked vegetable in various ways and rural people in India use dried shoots as fuel. It is good source of minerals and vitamins

and rich in total water soluble sugar, free reducing sugar and amide proteins etc.
However, in recent years, production of brinjal-eggplant in the Indian subcontinent has been seriously affected due to steady increase in insect pest infestation, especially the fruit and shoot borer; Leucinodes orbonalis (Guen.). The young larvae of fruit and shoot borer bore into petioles and midribs of large leaves and tender shoots causing shoot tip to wilt and later they bore into flower buds and fruits. The affected fruits lose their market value, over and above the considerable reduction in yield. In India it has been estimated that fruit and shoot borer cause damages to fruits ranging from 25.8-92.5 % and yield reduction from20.7 to 60% (W02007091277, http://v3.espacenet.com/). However, in some parts of the world its commercial production hampered by attack of Colorado potato beetle (CPB); Leptinotarsa decemlineata [Say.]; (W09807310Jhttp://v3.espacenet.com/).
Many important medicinal drugs are derived from plants in this family such as atropine and solasodine, the latter being the precursor of cortisone- type alkaloids used in family planning drugs (Hawkes.J.G., 2001). An invention related to the use of an eggplant extract to treat various medical conditions and diseases (US2007275109, http://v3 .espacenet.com/). Many pest and pathogen attack eggplant and reduce its yield and quality. Therefore, there is great concern in developing disease resistant varieties of brinjal-eggplant.
Wild Solarium species are valuable source for eggplant breeder as they are being source of resistance to many diseases and pests and also have many other traits of value in eggplant breeding; e.g. Solarium aethiopicum, Solarium torvum, Solarium sisymbrifolium and Solarium khasianum (Kashyap et.al.2001). In the conventional breeding in eggplant, developing interspecific hybrid verities has proved to be difficult due to phylogenetic distance. The success rate has been very low and results have only been achieved in few crosses (Daunay and Lester, 1998). Resistances have been identified in some Solarium species but have not yet been used, since they are

as yet either impossible (eg. Solarium torvum Sw., Solarium sisymbrifolium Lam.) or difficult (eg. Solarium macrocarpon L.) to transfer by sexual crossing (Daunay et al., 2001). An invention made on method of breeding new varieties of a first filial generation plant composed of new genomic constitution through inter specific hybridization in Brassica genus (United States Patent: 4102079, http://patft.uspto.gov/). A related invention on a novel plant species ssamchoo and to the breeding method there of; developed by in aneuploid breeding method between Chinese cabbage and cabbage and to the breeding method of ssamchoo (United States Patent: 6600092, http://patft. uspto.gov/).
The use of wild species related to Solanaceae crops has been of great value for the genetic improvement of many characters of interest in this group of economically important plants (Kalloo, 1988). Some distantly related species were highly resistant but are refractory for use in a conventional breeding programme, since they cannot be crossed with eggplant or produce only sterile hybrids (Robinson et al., 2001). Solarium torvum has attracted considerable attention from researchers because it carries many valuable pest and disease resistance genes (Sihachakr et al., 1994; Collonnier and Sihachakr, 1998). Hybrids of Solarium torvum and eggplant have been produced by interspecific pollination and embryo culture (McCammon and Hanma, 1983) and by protoplast fusion (Guri and Sink 1988a, Sihachakr et al. 1994; Collonier and Sihachakr 1998) but both the sexual and somatic hybrids are sterile and have not been used in eggplant breeding.
Crossability studies show that the cross compatibility between any two Solarium species is not a simple absolute characteristic; but rather there are degrees of success in each of a progression of stages after pollination i.e. seed normality, embryo viability, seedling viability, plant normality, pollen stainability and thus

fertility of interspecific progeny (Daunay et al. 2001). Baksh and Iqbal, 1979; Siddiqui and Khan, 1978; observed reduced pollen fertility in interspecific hybrids. Interspecific crosses between Solatium melongena and Solanum aculeatissimum', Solanum hispidum and Solanum khasianum are made only with difficulty and the resulting hybrids are sterile (Krishnappa and chennaveeraiah, 1965; Rao et al; 1979; Sharmaetal. 1980).
Phomopsis blight or fruit rot caused by Phomopsis vexans Sacc. and Syd. is the most serious and widespread brinjal-eggplant disease. It occurs on stems, leaves and fruits. However, in some parts of the world, commercial production of brinjal is frequently hampered by devastating infection by the phomopsis blight disease caused by Phomopsis vexans Sacc.and Syd. In the absence of an effective disease control programme, the Phomopsis vexans may cause total destruction of Brinjal-eggplant crop. Chemical pesticide application is costly to farmers and polluting the environment.
Since high level of resistance to phomopsis blight disease was not found with Solanum melongena; we screened distantly related Solanum torvum species for resistance. Also to asses the possibility of obtaining interspecific hybrids and introgressing genes from the wild species to the cultivated genetic background. Object of invention: l. To asses the possibilities of obtaining interspecific hybrids and introgressing genes
from wild species to the cultivated genetic backgrounds. 2.To develop disease resistant cultivars by introgressing the disease resistant genes from wild species Solanum torvum to the cultivated Solanum melongena L. background.

Summary of Invention:
With the background in mind; the present invention is aimed to provide a method for introgressing disease resistance gene(s) or other important traits and breaking incompatibility barriers between Solarium torvum and Solarium melongena. Another object of the invention is to provide strategy for screening the introgressed material for disease resistance and stabilization of the trait of importance.
A method of making interspecific F1 (first filial generation) seed comprising (a) crossing a cultivated species female parent with wild species Solanum torvum as a resistant donor to make interspecific F1 progeny seed (b) collecting said interspecific F1 progeny seed '(c) growing said interspecific F1 progeny (d) F1 progeny was observed to be highly sterile (e) backcrossing the F1 sterile progeny with both the parents (f) no backcross seed or embryos were achieved, thus backcrossing was failed (g) grafting cultivated species as a scion on wild species S. torvum as a rootstock (h) growing grafted cultivated species (i) growing grafted cultivated species female parent was crossed with wild parent Solanum torvum as a pollinator (j) few interspecific F1 progeny seeds were scored (k) simultaneously sterile F1progeny from (d) was crossed with grafted cultivated species as a pollinator (1) few Bc1F1 (1st backcross F1) progeny seeds were scored (m) growing BC1F1 progeny, increased pollen fertility was observed; but the fruits were berry type with rare seed set (n) backcrossing Bc1F2 progeny with cultivated species as a pollinator, collecting BC2F1 (Ilnd backcross ¥{) progeny seed (o) growing BC2F1 progeny and crossed with F1 progeny from (j) as a pollen parent, making DCHF1 i.e. double cross hybrid Fi. At the same time BC2Fi progeny selfed to make BC2F2 progeny seed (p) growing DCHF1progeny and BC2F2 progeny from (o) intercrossing both the progeny i.e. DCHF1 and BC2F2 reciprocally to maintain the resistant gene pool (q) growing the intercrossed progeny and screening for the elite introgressed segregants with improved fruit quality and resistance in segregating population (r) growing selected

elite introgressed segregants and selling (s) repeating the selling, characterization, evaluation and stabilization of traits of importance still homozygous plant progeny achieved (t) in each back cross and advance generations, population was tested for phomopsis blight disease reaction (u) at the end of this breeding cycle, three cultivable genotypes with good agronomic traits, fruit quality and resistance were scored. One genotype having round shape, green colour fruits with cut-leaf-lobed and another two having oval shape, green colour fruits with normal leaf and prickly and non-prickly traits.
Thus high fertility and crossability of material from interspecific crossing open the way to the utilization of Solarium torvum in brinjal-eggplant breeding programme. Detail description of invention:
The present invention provides a method for generating introgressed brinjal-egg plant cultivars resistant to traits of importance by breaking incompability barriers between two distantly related species, which is superior to other methods.
The breeding method which are composed of following steps of; obtaining the interspecific F1 between cultivated and wild species S. torvum, obtaining interspecific F1 on grafted cultivated species with wild species S torvum, obtaining BC1F1 progeny seed from sterile F1 progeny between cultivated species and wild species S. torvum with the pollination of grafted cultivated species; scoring DCHF1 i.e. double cross hybrid F1 by crossing BC2F1 progeny with F1 progeny (graft aided Fi); growing the DCHF1 progeny and BC2F2 progeny and attempted reciprocal intercrossing to maintain the resistant gene pool; growing and screening introgressed segregants for improved fruit quality, agronomical traits and resistance; repeating selfing of selected improved introgressed material; characterization, evaluation and stabilization of traits of importance still homozygous plant progeny achieved. At the end of breeding cycle, three elite cultivars having high fertility and crossability were

developed from interspecific crossing and this open the way to the utilization of S. torvum in brinjal-eggplant breeding programme.
Example: 1 (US6600092)
This example had been accomplished from 1998 -2006 using cultivated species and wild species Solarium torvum collected in 1995 while conducting exploration programme. In this breeding method cultivated species female parent crossed with wild species S. torvum as resistant donor to make interspecific F1 progeny seed. F1 progeny was observed to be highly sterile. Backcrossing above F1 sterile progeny with the grafted cultivated species and few Bc1F1 seed were scored. At the same time grafted cultivated species female parent was crossed with wild parent Solarium torvum as a pollinator and few interspecific F1 seed were scored. Growing BCiFi progeny, increased pollen fertility was observed, but the fruits were berry type with rare seed set. So back crossing Bc1F1 progeny with cultivated species as a pollinator to produce BC2F1 progeny seed. Crossing BC2F1 progeny female parent with F1 progeny (graft aided F1) to make DCFIF1 i.e. double cross hybrid F1. After that intercrossing DCHF1progeny and BC2F2 progeny (scored by selfing BC2F1 progeny) reciprocally to maintain resistant gene pool and scoring introgressed elite segregants. Repeating the selfing of elite introgressed segregants; characterization, evaluation and stabilization of traits of importance. Finally three elite cultivars with good agronomical traits, fruit quality and resistance were scored.
Example 2
Culturing the scored cultivars:
The cultivars obtained from the above example 1, were cultivated in different agro climatic regions in order to test their wider adaptability and recommended agronomical practices were carried out for the exploitation of material.

Example 3:
A specific traits of a cultivar (s):
The present example shows the characteristics of all the three developed cultivars viz. wider adaptability, improved traits including taste in comparison with prevailing cultivars of the region as control.

Principal coordinated analysis

The dendrogram derived from the similarity coefficient consist of two clusters. Cluster A was subdivided as sub-cluster Al, consist of cultivar 4,6,7,5,8 and cultivated parent. Where as sub-cluster A2 consist of cultivar 1,2,3,10 and 9. The cluster B of wild species S. torvum forming a separate lineage. In sub-cluster Al cultivar 6 & 7 as well as in sub-cluster A2 cultivar 1 & 2 were closely related genetically. The dendrogram generated, matrix comparison plot and principal coordinated analysis showed that the cultivars {S.melongena L.) and wild species S.torvum were genetically distinct.