| For integrating properties of fast growth and good timber characters in Section Populus and salt-alkali-resistance in Section Turanga of the genus Populus, techniques of pollen-tube pathway were systematically studied to introduce the exogenous DNA from P. euphratica to white poplars, including P. tomentosa×P. bolleana,P. alba×P. tomentosa and P. alba×P. glandulosa. These studies made some theoretical and technical bases for breeding of salt-resistant white poplars by pollen-tube pathway. Major results as follows:(1) Studies on the noncryogenic preservation of leaves tissue for DNA extraction of P. euphratica have been carried out, to settle the problems of preserving plant materials far away. The results indicated that, DNA from leaves stored in salica gel and SDS DNA extraction buffer for 20 days was good integrity and high purity . However, fresh leaves from branches cultured in water in winter or leaves preserved in salic gel within 10 days should be used and DNA extraction is employed by SDS method, to meet the needs in introduction of exogenous DNA via pollen-tube pathway. Furthermore, 2%β-Mercaptoethanol should be added to restrain brown of DNA.(2) Pollination tests of female catkins at different development stages show that, the receptivity and the optimal pollination stage of the stigmase are releated to morphological characteristics of the stigmas. At the optimal pollination stage, stigmas of P. tomentosa×P. bolleana catkins are evident,bright, slivers of stigmas flat about 180°; P. alba×P. glandulosa bracts expand, stigmas are evident,bright, and slivers of stigmas flat 180°; P. alba×P. tomentosa bracts expand too, stigmas are evident,bright, and slivers of stigmas splay about 60°～90°. Pollination at the the optimal stage could help accurate the starting introduction time. According to breeding of crops, when introduction of exogenous DNA into white poplars via pollen-tube pathway is carried out, the effective treatment time of P. tomentosa×P. bolleana and P. alba×P. glandulosa is both 36～72h after pollination, and P. alba×P. tomentosa is 36～60h after pollination.(3) Transformation of exogenous DNA of P. euphratica via pollen-tube pathway and combination of the introduction and chromosome doubling of embryo sac were studied at different time after pollination and treated for different hours, with P. tomentosa×P. bolleana,P. alba×P. bolleana and P. alba×P. glandulosa as female parents, P. tomentosa and P. bolleana as male parents. A total of 14,721 seeds were harvested and 883 offspring were conserved in field. Seed sets decreased after transformation of exogenous DNA via pollen-tube pathway, which may be resulted from mechanical damage when stigmas cut or poison of retained organic reagents in DNA liquor. Analysis of phenotype characteristics showed that most of offspring were similar to parents and only 2 ones presented variation of leaves similar to lanceolate leaves of P. euphratica. In the progeny, no polyploid was detected.(4) 70 offspring from treatments started from 24 to 54 hours after pollination and treated for 2 to 18 hours were detected as putative transformed plants at molecular level by amplified fragment length polymophysm (AFLP). Among them, there were 62 individuls with new bands of type A (presenting only in treated progenies but not parents,CK and dornor) and another 8 ones possessing new bands of type B (presenting only in treated progenies and dornor but not parents,CK). However, no molecular variation was found in those 2 offspring with phenotypical variation. The sequencing results and alignment results of couples specific bands showed that the results of AFLP might be"false positive", so it was not reliable to detect transformed plants with exogenous genomic DNA via pollen-tube pathway only relying on specific bands detected in primary molecular marker. The result showed that individual MM10-2 was hybrid progeny which was integrated by homologous recombination of exogenous P. euphratica partial DNA.
|
PDF Full Text Request