Genetical Analysis Of Intertribal Hybrids Of Brassica Napus With Lesquerella Fendleri And Isatis Indigotica

Interspecific / intergeneric hybridization plays an important role in crop genetic improvements. Extensive ranges of wild relatives for Brassica crops provide rich germplasm with many useful nuclear and cytoplasmic genes for oilseed breeding. But it is difficult to use the wild relatives in agriculture directly because of low yield. The transfer of desired traits from wild germplasm to cultivated crops by sexual/ somatic hybridization has been realized successfully in plant breeding. Lesquerella fendleri Gray (Wats.) (2n=12) which belongs to the tribe Drabae of the family Brassicaceae is an annual native to the arid and semiarid regions of Southwestern America and has been proposed as an important industrial oil crop. Its meal after oil extraction is rich in protein and has an excellent distribution of amino acids being particularly high in lysine. L. fendleri is one new valuable genetic resource for the rapeseed breeding of industrial purpose as it possesses favorable fatty acid compositions containing high amounts of hydroxy fatty acids, about 60% lesqueroloc acid (20:1^{â–³11,14-OH}). Another important relative, I. indigotica (2n= 14) belonging to the tribe Lepidieae of Brassicaceae has been used from ancient times as indigo-producing plant and particularly medicinal plant. The root of I. indigotica (Radix Isatidis) is a commonly used traditional Chinese medicine to remove heat and eliminate toxin, to reduce heat in blood, and to soothe the sore throat. It also shows high degree of resistance to tobacco mosaic virus (TMV) and Sclerotinia sclerotiorum. In the present study, intertribal hybrids between Brassica napus L. (2n=38) and Lesquerella fendleri, I. indigotica were produced by sexual and somatic hybridization and characterized for their morphology, cytology and genomic components. The main results were as follows:1. B. napus×L. fendleri. Twenty-seven F₁ plants were produced by the seeds harvested (Nos. 5, 6, 9, 17, 20, 21, 27) and the rescued immature embryos (the remaining 20 plants). F₁ plants and their progenies mainly resembled female B. napus parents, while certain characters of L. fendleri were expressed in some plants, such as longer flowering period, basal clustering stems and particularly the glutinous layer on seed coats related to drought tolerance. These F₁ plants were cytologically classified into five types: type I(16 plants) had 2n=38, typeâ…¡(2) had 2n=38-42, typeâ…¢(3) had 2n=31-38, typeâ…£(5) had 2n=25-31, and typeâ…¤(1) had 2n=19-22. Some hybrids and their progenies were mixoploids in nature with only 1-2 chromosomes or some chromosomal fragments of I. fendleri included in their cells. AFLP (Amplified fragments length polymorphism) analysis revealed that bands absent in B. napus, novel for two parents and specific for L. fendleri appeared in all F₁ plants and their progenies. Some progenies had the modified fatty acid profiles with higher levels of linoleic, linolenic, eicosanoic and erucic acids than those of B. napus parents. One reason leading to the change of fatty acid compositions might be attributed to LFAH12 introgression, an oleate 12-hydroxylase gene from L. fendleri which had both hydroxylase and desaturase activities.2. Somatic hybridizations between B. napus and I. indigotica2.1 Plant regeneration. Factors which affected regeneration frequency of protoplasts were analyzed, such as the explant states, enzyme combinations, AgNO₃ concentrations and the proportion and sorts of hormone. A total of 237 calli and 15 plantles were produced among three combinations of I. indigotica with three cultivars Huashuang No. 3, Zhongyou821 and Oro, the regeneration frequency being 6.7%.2.2 Morphology of somatic hybrids and progenies. At the early growth stage, the leaves of the hybrids were more darkly greener and thicker than those of B. napus parents and covered with more heavily waxen powder. The hybrids were shorter flowered later than B. napus parents. Their flowers had poorly-developed stamens with only 3-5 ones in most flowers and were sterile for male. Furthermore, the anthers were deeply brown and had a special aroma, as those of I. indigotica. The hybrids were partially fertile for female and produced some seeds after pollinated by B. napus parents. The BC₁ plants showed predominantly the morphology of B. napus and expressed a few traits from I. indigotica, such as heavy waxen powder, dark green and thick leaves and the same smell of flowers.2.3 Cytology of somatic hybrids and progenies. The hybrids S2 (symmetric fusion) and AS1 (asymmetric fusion) had the expected 2n=52 in ovary cells and produced pollen mother cells (PMCs) mainly with 2611 at diakinesis and 26: 26 segregation at anaphase I (AI), with 14 chromosomes strongly labeled by the I. indigotica probe. The plants AS4, AS6 and AS7 were mixoploids (2n=48-62). The hybrid AS 12 had 2n=66 in ovary cells. The BC₁ plants had 2n=45 in all the ovary cells and their PMCs mainly showed the AI segregations 22:23, 21:24, 20:25, and 7 chromosomes from I. indigotica presented autosyndetic (within genome) or and allosyndetic (among genomes) pairings.2.4 Nuclear and cytoplasmic genomic analysis. AFLP and SSR analysis showed all the hybrids had mainly the DNA banding pattern from the addition of two parents plus some alterations, such as deleted bands in parents and novel bands. Universal primers analysis on organelle DNA showed that chloroplast DNA in AS8, AS9, AS 10 and AS 11 were from I. indigotica and mitochondrial DNA in all the hybrids were from B. napus without organelle recombination.