| Bambusa multiplex (Gramineae, Bambusoideae), a sympodial bamboo species with pachymorph rhizome was chosen as material for genetic improvement research, considering its wide distribution and strong stress-resistance. In this study, phenotypic traits of five wild populations of B. multiplex in China were investigated and analyzed, genetic diversity was measured by AFLP markers, flowering habit and crossing test were conducted, and callus induction and plantlet regeneration of different explant types was experimented, the results are as follows:1. Phenotypic variation of B. multiplex were conducted by using five geographical populations of Wen Ling, Long Shan, Lu Zhou, Ren Hua and Chang Ting, China, 20 culms of each were cut down for samples. Total 19 phenotypic traits were measured and analyzed, including diameter of breast height (dbh), diameter of culm base (dcb), dbh/dcb, culm length (cl), full length (fl), cl/fl, culm fresh weight (cfw), full fresh weight (ffw), cfw/ffw, number of culm internode (nci), mumber of full internode (nfi), nci/nfi, maximum internode length (mil), number of internodes under branching (nib), height under branching (hb), angle of branching (ab), leaf length (ll), leaf width (lw) and ratio of ll/lw. The results showed that abundant variation existed within and among populations, 14 traits of Lu Zhou population toped the five and 11 traits of Ren Hua population bottomed. 7, 5, 4, 2 and 1 traits in Long Shan, Chang Ting, Ren Hua, Lu Zhou and Wen Ling had the largest variance coefficient, respectively. Correlation analysis demonstrated that difference at 1% to 5% significant level existed between 14 phenotypic traits including dbh, dcb, etc. While five phenotypic traits of number of nci, ratio of nci/nfci ,ll, lw and ratio of ll/lw. Some traits exhibited correlation to geographical and ecological factors, for example, significant positive correlation of cfw/ffw to latitude, ll/lw to altitude, and negative correlation of ll to longitude, ll/lw to longitude, etc. 3 principal components were obtained after analysis by using 19 phenotypic traits with a cumulative contribution of 97.44%. Index of principal components were introduce into the original values of each population and a cluster analysis was developed, five populations were divided into 3 groups by phenotypic traits, Wen Ling and Chang Ting joined together, Long Shan and Ren Hua joined, Chang Ting was the third group alone. 2. 10 primer combinations of AFLP were screened out for genetic analysis of the above five populations of B.multiplex, each with 30 samples. Total 939 loci were detected with a polymorphic percentage of 71.99%. In each population, polymorphic loci (A) varied from 106-464 with an average of 255.8; polymorphic percentage (P) changed from 11.29%-49.41% with an average of 27.24%; Observed alleles number (na) was 1.1129-1.4941; Effective alleles number (ne) was 1.0713-1.3147; Nei's gene diversity (h) was 0.0406-0.1794; Shannon's Information index (I) 0.0601-0.2656. All of these index showed a tendency of Chang Ting>Ren Huap> Wen Ling>Lu Zhou>Long Shan in five B.multiplex populations, indicates that a rich variations existed in Chang Ting while a low genetic diversity in Wen Ling. Coefficient of genetic differentiation of B.multiplex (Gst)was 0.4393, which means a 43.93% genetic variation occurred between populations while 56.07% genetic variation in population; gene flow (Nm) among populations was 0.6383. Nei's genetic identities in five B.multiplex populations were between 0.8165-0.9675, and genetic distances were between 0.0330-0.2027. Five populations showed a molecular phylogeny of Luzhou and Long Shan were clustered together firstly, then Wen Ling joined, and the Chang Ting was the last.3. Studies on flowering biology and crossing of B.multiplex showed that it's inflorescences usually formed in April, spikelets opens in the morning and closes at 14:00-15:00 p.m.. Pollen germinated percentage can be over 50% at the time that the elongation growth finishes of filament. Stigma receptivity is the best when glumes open during 9:00-12:00 a.m.. Seeds from selfing of B.multiplex and B.multiplex×Dendroclamus latiflorus doesn't show apparent difference, they were long oval, inconspicuous ventral furrow, browning yellow when mature with 0.82-1.10 cm long, wideth0.26-0.28 cm wide ,weight 3.32 g per 100 seeds. Progenies showed significant difference from selfing and crossing at four month's age, putative hybrid showed a more larger leaf than that of selfed ones. At this age, the leaf length, leaf width, leaf length-width ratio, ground diameter, shoot numbers, leaf vein numbers, leaf shapes, leaf sheath height, leaf sheath hair, ligules, auricle of putative hybrids shows a middle value between the paternal and maternal selfed progenies. AFLP identification of hybrids were conducted, paternal particular loci changed from 21.97%-28.10% in 10 hybrids, and non-parental loci were 1.88%-4.05%, gave a molecular confirmation of true hybridity. Sheath of hybrids shows segregation in cilia at fifteen month's age, one is the paternal ciliate and the other shows the maternal glabrous. Such a study make a base for elite hybrids selection and genetic analysis of important taxonomic taits of sheath ligule, auricle, blade and spot, etc..4. Embryogenic callus was initiated and plant regeneration has been achieved by culturing of spikelets and seed embryo explants of B. multiplex. 87.30% and 76.27% callus were induced 475 mg.L-1+ NH4NO3 825 mg.L-1+ MgSO4·7H2O 185 mg.L-1 + KH2PO4340 mg.L-1 + CaCl2·7H2O 440 mg.L-1+MS basal micronutrients + MS Iron + MS vitamins +1.0 mg.L-1 proline+30 g.L-1 maltose+8 g.L-1carrageenan+4 mg.L-1 2,4-D, callus can proliferate under stress of 0.0 g.L-1NaCl, 75 mg.L-1 Hygromycin was useful in callus resistance selection. A good suspension culture system of callus was an inoculation density of 4.0%, rotation speed was 120 r/min, 2,4-D concentration was 4~6 mg/L, and subculture every 7 days.Plantlet regenerated by two stage of pre-differentiation and germination, after a 7 d pre-differentiation on MS supplemented with 30 g·L-1 sucrose, 10 g·L-1 Carrageenan and 4 mg·L-1 kinetin (KT), and a 14 d auxin-free germination culture on MS, only a few spikelet calli germinated; whereas embryo calli regenerated by up to 80.0%; about 8% plantlets were albinal, and mosaic plantlets were occasionally found. Optimal pre-differentiation medium for seeds embryoids was MS supplemented with 3 mg·L-1 6-benzylaminopurine (6-BA) and 3 mg·L-1 KT; plantlets rooted in MS supplemented with 2 mg·L-1 naphthaleneacetic acid (NAA). Rooted plantlets transferred to soil with over 70% success. Such an effective regeneration system of B.multiplex made it possible for genetic transformation mediated by agrobacterium tumefaciens. |
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