Production And Genetic Analysis Of Intertribal Hybrids Between Medicinal Plant Isatis Indigotica And Cultivated Brassiceae Species

Wide hybridization plays an important role in crop genetic improvements.Related or distant species of cultivated crops contain a large reservoir of genes covering a variety of desirable traits like resistance to biotic or abiotic stresses.Gene transfer from these species to cultivated crops via wide hybridization including sexual and somatic hybridization could create novel plant forms and improve qualities of crops,also broaden genetic basis of cultivated crops and increase their genetic variability in breeding programes.Isatis indigotica Fort.(Chinese woad or tein-cheing,2n=14,II),belonging to the Isatideae tribe of the Brassicaceae family,is a dye crop with a greater indigo-production capacity.More importantly,as a traditional Chinese medicine,Ⅰ. indigotica contains compounds which may cure many diseases caused by bacteria and viruses and increase the protective immune response.Additionally,Ⅰ.indigotica also shows resistance to tobacco mosaic virus(TMV) and stem rot(Sclerotinia sclerotiorum), the most serious disease of rapeseed in China.So these available traits may be very beneficial for improving quality of cultivated crops.In the present study,intertribal sexual partial hybrids of Brassica rapa,B.napus with I.indigotica as pollen parent and intertribal somatic hybrids or hybrid calli between B.rapa,Raphanus sativus,B.oleracea L.var.alboglabra Bailey and I.indigotica were produced and characterized for their morphology,cytology and genomic components.The main results were as follows.1.Sexual hybridizations between B.rapa,B.napus and I.indigoticaIn B.rapa×1.indigotica combination,many hybrid plants were obtained from the mature seeds harvested and then exhibited different morphological traits.However, majority of them did not survive and only three plants grew to maturity.These 3 hybrids showed poor growth and much smaller stature than the two parents,but had some morphological traits and chemical composition of I.indigotica.One plant had 2n=10,the haploid chromosome number of B.rapa and was absolutely sterile.The other two plants had 20 and 22 somatic chromosomes and were male sterile but produced seeds following pollinations with B.rapa.All backcross progenies over several generations maintained a B.rapa-type phenotype and also displayed some variations in morphological characters and fatty acid compositions.They were all 2n=20 and showed good seed-set.The hybrid with 2n=22 produced some progeny plants with 2n=21 and 2n=22.Genomic in situ hybridization(GISH) detected two chromosomes of I.indigotica in the hybrid with 2n=22 but none in the one with 2n=20.AFLP bands specific for I.indigotica,novel for two parents or absent in B.rapa were detected in the two hybrids and their progenies.These progeny plants were novel B.rapa types with altered genomic constitution or alien additions.The hybrids and progenies were the outcome of the chromosome elimination, addition and introgression.In B.napus×I.indigotica combination,45 mature seeds and 85 immature embryos were produced,but only five hybrids(H1-H5) were distinguished morphologically from female B.napus parents by showing low fertility and some characters of I.indigotica,and also having different chromosome numbers.H1-H4 had similar but variable chromosome numbers in their somatic and meiotic cells(2n=25-30),H5 had 2n=19,the same number as the haploid of B.napus.GISH analysis detected one I.indigotica chromosome and one or two chromosome terminal fragments in H1,H5.New B.napus types with phenotypic and genomic alterations were produced by H1 after pollination by B.napus parent and selfing for several generations,and by H5 after selfing.The B.rapa-type progeny(2n=20) was derived from H1 after pollination by I.indigotica twice,showing that the A-genome chromosomes in B.napus were preferentially maintained and those from C-genome were inclined to be lost under the induction of alien chromosome elimination in wide cross with I.indigotica.The reasons for the formation of the partial hybrids with unexpected chromosomal complements and for the genome-specific chromosome stability in Brassica allopolyploids were discussed.2.Somatic hybridizations between B.rapa,R.sativus,B.alboglabra and I.indigotieaIn B.rapa+ I.indigotica combination,five mature hybrids were produced during symmetric fusions of mesophyll protoplasts.They were morphologically intermediate but showed some differences in phenotypic traits and fertility,two were partially fertile. Cytological and GISH investigations revealed that these hybrids had 2n=48 with AAIIII complement and their pollen mother cells(PMCs) showed normal pairing of 24 bivalents and mainly equal segregation 24:24,but meiotic abnormalities of lagging chromosomes and micronuclei appeared frequently during second divisions.AFLP analysis showed that all of these hybrids had mainly the DNA banding pattern from the addition of two parents plus some alterations.Some hybrids should be used for the genetic improvement of B. rapa and the dye and medicinal plant.In R.sativus+I.indigotica combination,one mature hybrid established through symmetric fusions of mesophyll protoplasts had intermediate morphology but was totally sterile.It had the expected chromosome number(2n=32,RRI I) and parental chromosomes were distinguished by GISH analysis,and these chromosomes were paired as 16 bivalents in PMCs at diakinesis and mainly segregated equally as 16:16 at anaphase I(A I),but the meiotic disturbance presented obviously in second division.In B.alboglabra+I.indigotica combination,intertribal hybrid calli were induced and produced by using polyethylene glycol(PEG,3350) and DMSO.The randomly selected 5 hybrid calli and their parents were subjected to SRAP analysis by using 9 pairs of primers randomly selected,and the results showed that the calli had mainly the DNA banding pattern from the addition of two parents,but also had a few novel bands for two parents.SRAP clustering analysis showed that their genomic compositions were similar but closer to I.indigotica than to B.alboglabra.