全 文 ：广 西 植 物 Guihaia 29(2)：192— 197 2009年 3月
不同居群白木香的染色体研究
申彦晶1，焦旭雯1，赵树进 ，2
(1．华南理工大学 生物科学与工程学院，广州 510641；2．广州军区广州总医院 药学部，广州 510010)
摘 要：采用常规压片法和改良BSG法对 3个居群白木香的染色体核型及 Giemsa C-带带型进行研究。结果表
明：3个居群白木香的核型均属 2B类型，其中广西居群白木香的核型公式为 2n=16—4m+8sm+4st；其他两个
居群白木香的核型公式为 2n=16=6m+6sm+4st，居群间核型变异不明显。白木香的 C带带型为 CIT型，具有
着丝粒带、中间带、端带和全带。3个居群白木香 C带的分布、数 目和类型不完全一样，出现了带型的多态性。
关键词：白木香 ；核型；Giemsa C带
中图分类号 ：Q343．22 文献标识码：A 文章编号 ：1000—3142(2009)02—0192—06
Chromosomal studies on populations
of Aquilaria sinensis
SHEN Yan-Jing1，J IAO Xu-Wen1，ZHAO Shu-Jin1，2
(1．College of Bioscience and Engineering，South(7hina(niversity of Technology，Guangzhou 510640。China；2．Department
of Medicament，General Hospital o／Cruang．zhou Military Command，Guangzhou 510010，China)
Abstract：For the first time，a conventional anal ysis of chromosome karyotype and Giemsa C-banding was carried out
in three populations of Aquilaria sinensis．Three populations showed same chromosome number of 2n=2x=1 6 and
the asymmetry of the karyotype was categorized as type 2B．The variance of karyotype was not obvious in three pop—
ulations，only karyotypical formula(2n一 16—4m+8sm+4st)in one population was different from the other two(2n
一 16—6m+ 6sm+ 4st)．The Giemsa C—banding pattern of A．sinensis was CIT pattern．W ithin Giemsa C-banding
pattern of A．sinensis there were centromeric bands，intercalary bands，telomeric bands and whole bands．The distributed
position，numbers and types of the C-banding oil the chromosome of A．sinensis had shown the clear polymorphisms．
Key words：Aquilaria sinensis；karyotyp e；Giemsa C-banding
A．sinensis is almost distributed in Guangdong，
1 Introduction
Aquilaria sinensis(Thymelaeaceae)is one of very
few species of tropical trees and is the source of agar—
wood(Ng et a1．，1997)，one of the most highly valua—
ble forest products currently traded internationally．
Agarwood(also known as aloeswood，eaglewood and
gaharu，among many other common names)is a fra—
grant wood that has beel1 traded since biblical times for
use in religious functions and for medicinal and aromat—
ic preparations(Barden et c ．，2000)．
Hainan and Guangxi provinces in China． Since the
15th Century，agarwood has been collected and used as
a drug in China． Studies revealed that agarwood has
remarkable anticancer activity (Gunasekera et a1．，
1981)．Benzene extractable compounds possess potenl
central nervous system antidepression activities(Oku—
gawa et a1．，1993，1996)，and agarwood is considered
as new promising nervous system drug(Chen，1 999)．
High consumer demand，particularly from Middle East—
ern and Asian markets，combined with decreasing sup—
ply has pushed prices progressively higher to the extent
Accepted date：2007—06—25 Received date：2008—05—22
Foundation item：Supported by Natural Science Foundation of Guangdong Pro~nce(06019716)
Biography：SHEN Yan—Jing(1980)，born in Henan Province，PH．D，research in Biopharmary，Email：shyanjing@tom．eom
Author for corresp0ndence
2期 申彦晶等：不同居群白木香的染色体研究 193
that top grade agarwood can sel for over USD 1 0 000／
kg in end—use markets(Barden et a1．，2000)．
Previous studies of A．sinensis have mostly fo—
cused on its tissue culture，chemical composition and
pharmacologica1 action of agarwood．However，its cy—
togenetic data remain unknown． In this article we
studied the chromosome karyotype and Giemsa C-ban—
ding pattern of A．sinensis occurring in different popu—
lations，which offered the evidence for further research
of identification，breeding and evolution．
2 Materials and methods
2．1 Plant material
Seeds of A．sinensis were from Nanning (Guan—
gxi)，Maoming(Guangdong)，Tunchang (Hainan)and
identified by Xing Fu—W u，professor of South China
Botanical Garden in Guangzhou．
2．2 Karyotype analysis
Roots were colected from potted plants of selected
genotypes kept in a shade house．Root tips were treated
with 0．002 mol／L 8一hydroxyquinoline solution for 2 h at
18—2O℃ ，and fixed in Carnoy’s I fixative(etlano1：
acetic acid一3：1 v／v)for 2—24 h．Samples were
hydrolysed in 1 mol／L HC1 at 60℃ for 8—10 min。and
digested with 2．5 cellulase enzyme for 30 min at 37
℃。then squashed in a drop of Carbol fuchsin on a mi—
croscope slide．w th the cover slip in place。the slide was
heated and then pressed firmly to flatten cels．All ob—
servations were made under oil immersion objective lens
using a Olympus(BX41)microscope． Only metaphase
cels，in which individual chromosomes were clearly dis—
tinguishable，were used for making counts and> 10 di—
vidial cels were counted for each sample to determine
the chromosome number．Photographs of the 3— 5 best
individual cels were enlarged for karyotyping．
Take photos after the chromosome were properly
stained and select clear photos for analysis． A1 least
three root tip cells per individual were used to deter—
mine the karyotype of somatic chromosomes at meta—
phase．We measured the haploid absolute length and
the sym metry index，which was calculated from the ra—
tio between the sizes of the largest and the smallest
chromosomes(Stebbins，1971)．Terminology of chro—
mosome morphology based on the position of a centro—
mere followed Levan (1964)． For comparison among
different karyotypes at mitotic metaphase，a karyotype
formula was used．
2．3 Giemsa C-ban ding
The root tips were pretreated with 0．002 mol／L
8一hydroxyquinoline solution for 2 h at 18— 20℃ and
then fixed as above．They were rinsed in distilled wa—
ter and then hydrolysed in 0．2 mol／L HC1 at 60℃ for
8— 10 min，and digested with 2 cellulase enzym e for
30 min at 37℃ ．They were subsequently squashed in
the 45 acetic acid．The cover slips were removed by
freezing in the liquid nitrogen and put in the pure etha—
nol for l h，then dried in the air for 2days．The slides
were incubated for 7 minutes in the saturated Ba
(OH)2 at room temperature and washed them quickly
with distilled water．Then the slides were incubated in
2×SSC at 60℃ for lh and be stained with 2 Giem—
sa(diluted in distilled water)for about 60— 9O min．
Select the photos whose chromosomes are in—
tegrate and bands are clear to analyse the chromosomal
bands． Refert the Karyotyping and further confirm
them by Scanner and Microsoft photo editor，then syn—
thesize the C-banding pattern．
3 Result
3．1 Karyotype analysis of A．sinensis
Somatic chromosome numbers for A．sinensis in
three populations were all 2n=16(Plate I：1，2)and the
basic chromosome number were x= 8．
For the three populations specimens examined，the
karyotype formula，chromosome length，genome length
and asymmetry indexes are listed in Ta ble 1． The
karyotypical formula of three population was 2n=16=
6m+6sm+4st)and they have same karyotype charac—
ters．They comprised 6 metacentric chromosome pairS
and 6 submetacentrie chromosome pairs and 4 subter—
minal chromosome pairs．
Even though the species studied were found to be
homogeneous in karyotype form ula，intra species varia—
tion related to chromosome size and genome length
were observed．Population P3 had the biggest chromo—
some length range(2．76—6．96 Urn)，P1 3n intermediate
194 广 西 植 物 29卷
Plate I 1．Micrographs of chromosomes of A．⋯lensis(×1 000)，2n=16，Scale bar=5 f』Ill；2．Chromosomes karyotype
0f A． inensis；(Scale bar=5 m)；3．Idiograms of somatic metaphase chromosomes of A．sinensis．
size(2．87—6．96 m)and P2 had the smalest(2．97
— 7．01 m)．Intergeneric variation in genome length
was also observed． Mean genome 1ength was 41．02
m for P1，41．83 m for P2 and 40．08 gm for P3．
3．2 Gimesa C-banding of A．sinensis
Five pairs chromosomes have centromeric C-bands
of population Pl(PlateⅡ)．Chromosome 2 and 6 have
telomere bands on both the short and the long arms．
Chromosome 4 and 5 have telomere bands on the short
arms and intercalary bands on the 1ong anTiS．Chromo—
some 3 has a completely heterochromatie short arm．
The Giemsa C-banding pattern of P1 iS 2n=2x= l 6—
4C+2T+6C +2TI++2T I+．
For population P2(Plate I)，5 pairs chromosomes
have centromeric CHbands，like population P1． Chro—
mosome 1 has three C-bands，including tWO telomere
bands and one intercalary band on the long arm．Chro—
mosome 3 and 7 have centromeric bands and intercalary
bands on the 1ong arms．Chromosome 4 has telomere
bands on both the short and the long arm．The C-ban—
ding pattern is 2n=2x=16—4C+2T+2CT+ 2CI++
2CT+I _{一4T I+ Like Pl and P2，population P3 also
have 5 pairs chromosome with centromeric C-band
(Plate ID．Chromosome 1 and 2 have completely het—
erochromatic short arrfl and intercalary band on the
1ong arms，moreover，chromosome has one telomere
band．Chromosome 3 and 5 have intercalary bands on
tt1e long arms．Chromosome 4 has intercalary bands on
both the short and the long arms．Chromosome 7 has
telomere bands on both the short and the long arms．
The Giemsa C-banding pattern of P3 is 2n=2x=16=
4C+2I+2T+2CI++2CI+T+ 2CI+T++ 2I+T’卜．
2期 申彦晶等 ：不同居群白木香的染色体研究 l95
Tabl e l Parameters of chromosomes in
the populations of A．sinensis
4 Discussion
Three populations of A．sinensis examined in this
study have a consistent somatic chromosome number of
2n一 1 6．The ratio of the longest to the shortest chro—
mosome in all three populations exceeded 2．00，indica—
ting that the species we studied have a relatively high
interchromosomal asymmetry．The chromosome num—
ber of A．sinensis agreed with that reported by De—
benath et a1．(1995) for Aquilaria agallocha from
Sipahijala forest，India．But some differences still lies
in these two species．The karyotype of A．agallocha is
symmetric with 5 metacentric pairs and 3 sub—。metacen—
tric pairs，one of which has secondary constriction．The
chromosome type of A．sinensis in three populations
belonged to category 2B which was not so symmetric
as A．agallocha．In these populations，there are more
sub—metacentric pairs than A．agallocha and no sec—
ondary constriction occurred．A．sinensis comprised 2
pairs subterminal chromosome which didn’t exisit in
A．agallocha．And the chromosome researches on oth—
er species of genus Aquilaria have not recorded．
According to the classification proposed by Steb—
bins，the karyotypes of the three populations can be in
serted within category 2B (categories range from 1 A
(most symmetrica1)to 4C)，a more asymmetry karyo—
typype．Stebbins regarded that the foundational trend
of the karyotype evolution is from symmetry to asym—
metry，during the systematic evolution，the majority of
ancient or primitive plants have more symmetric karyo—
type．Whereas，asymmetric karyotype is often seen in
the plants which are derivative or in the advanced stage
of evolution．So A．sinensis can be considered as ad—
vanced as for their karyotype．
Table 2 Comparison of karyotypes among
populations in A．sinensis
The C-bands of A．sinensis in three populations al
showed centromeric bands，telomere bands and interca—
lary bands distributing among various chromosomes．
But the number and position of C-bands among popu—
lations were different．C-banding showed conspicuous
bands in numerous chromosome pairs，located at cen—
tromeric positions，with some pairs also characterized
by C-bands at the telomeric position on the short arms
or heterochromatic short arms．Giemsa G bands of A．
sinensis had not only centromeric bands but also te一
1omere bands and intercalary bands，furthermore，bands
patter variety didn’t happen only on the short arms．
C_bands differentiati0n of A．sinensis chromosome oc—
curred both short arms and long arms，which indicated
A．sinensis had advanced evolution leve1．It was sugges—
ted that C-bands had polym orphism among populations，
which can be used as a genetic index to analysis relations
among genus Aquilaria with high reliability．
The chromosomal karyotype displays the charac—
ters of the species at the chromosome set level，the
chromosomal C-banding shows the spread of the con—
stitutive heterochromatin in the chromosome(Jellen&
l96 广 西 植 物 29卷
PlateⅡ 4．Chmmosomes c anding。f A． inensi (Seale bar=5 m)；5．Idiograms。f sumatic metaphase chromoson1es c—banding of A-sinensis．
Ladizinsky，2000)． The pattern of heterochromatin
distribution has been determined for a large number of
species and populations in studies that have investiga—
ted the possible polymorphisms and／or variations，in an
attempt tO understand phyletic interrelationships(Lou—
renc0 fⅡZ．，1 9 98)．Because the polymorphisms of the
chromosomal bands endows the specific recognition
n1arker to the chromosomes and offer the evidences f0r
genetic analysis among the different or the same species，
so h can be used as a genetic marker tO study the chro—
mosomal operation species evolution and the relation of
diferent species genome(Hiseyin& Sema ，2005)．
The res ts of this study help to clarify the chro—
mosoH1a1 differences among populations of A．sinensis
with regard tO the amount and location of heterochroma～
ti n』()T1ce these banding patterns have been established，
in other species a more detailed analysis of the evolution～
ary relationships of the species will be possible．
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Stafleu，556 P．1967；Utrecht：International Bureau for Plant Taxonomy and Nomenclature；Taxonomic Literature，2nd
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2，H—Le，991 P，1979；3，Lh-O，980 P，1981；4，P-Sak，1214 P，1983；5，Sal-Ste，1066 P，1985；6，Sti-Vuy，926 P，1986；7，
W-Z，653 P，1988；Supplement，Frans A．Stafleu&Erik A．Mennega．Konigstein：Koeltz Scientific Books，I．A-Ba。453
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2000；Laurence J．Dorr& Dan H．Nicolson，K6nigstein：Koeltz Scientific Books，Ⅶ．F-Frer，470 P，2008，& Ⅷ．Fres-G，
558 P，2008．
本书是植物分类文献目录中最重要的工具书，是考证各类文献与时间(跨度从 1753到 1940年)的权威著作。
TL-2共 l5卷 ll 318页，包括 9 072位作者的文献 37 600条，编写时间前后达 32年之久。无论是收集的内容还
是覆盖的范围，该书都可以说是当今世界植物分类学界的宏伟巨作；不仅是对古典植物分类学文献，同时也包括
当代的各种文献；不仅收录范围包括广义的植物学(藻类、真菌、地衣、苔藓、蕨类、种子植物)，同时兼收古植物学
以及少数生物学的内容。本书的编排方式以作者为序，按姓氏字母排列。每个作者都有详细的全名与缩写、生平、传记、工作单
位、研究过的标本以及模式标本存放地点、出版物等介绍；同时还列举有关的参考文献以便读者进一步考证或获得更多的知识。
每个作者 的论著按年代排列，包括论著的全称与缩写 、出版时间 、地点、卷册 ，页码 ，图表 ，版次 ，以及相关的评论等。该书第一版仅
一 本，收录的论著 1 453种．共 556页。编者Frans A．Stafleu(1921～1997)是荷兰人，不仅是 International Association for Plant Tax—
onomy(IAPT)的秘书而且长期工作于植物分类学界著名的 Utrecht University，是世界上著名植物学文献、历史和命名方面的权
威 ；一人仅用三年的时间(1965～1967)完成这样的巨作 ，除了长期积累的资料外 ，更重要 的是知识 、智 慧和阅历 。第 二版 Frans A．
Stafleu联合美国斯密森研究院国家自然历史博物馆(Natinal Museum of Natural History，Smithsonian Institution)刚卸任的主任
Richard S．Cowan(1921～l998)并扩充 内容(参见 Taxon 28(1，2／3)，77—86，1979)，两人历时 12年之久 (1973年 l1月到 1985年 10
月)完成近 7 000页的 7卷本，其内容是第一版的 lO多倍，论著达 18 785种；最终使该书成为世界植物分类学家必不可少的工具。
然而由于第二版从第二卷即1976年开始得到美国国家自然科学基金(National Science Foundation)的资助，所以增加很多内容，但
先行完成的第二版第一卷(即作者 A到 G)是在没有经费的情况下进行的，和第二卷以后各卷相比需要补充。第二版完成后，
Richard S．Cowan由于已经退休而不参加余下的工作，于是Frans A．Stafleu又联合 Utrecht University的 Erik A．Mennega(1923～
1998)~~第二版的第一卷进行补订。遗憾地是补订工作由于两位作者 1997年和 1998年先后故去，只完成 A到E(其中补编第V和
Ⅵ卷的内容 D-E由德国柏林植物园的 Norbert Kilian和 Ralf Hand在前人的手稿基础上编辑完成)，尽管他们当初希望能够完成第
一 卷的全部 A到 G。第二版第一卷的补编工作任务相当大 ，同时也增加很多内容 ，到补编Ⅵ时 已经收载论著已达 33 658种 。幸亏
美国斯密森研究院 Dan H_Nicolson联合 Laurence J．Dorr接过续编，完成补编余下的两册(F-Frer和 Fres-G)并于最近 出版。大多
数中国植物学工作者都知道 Dan H．Nicolson先生，不仅因为他是当代世界上植物分类学领域的著名权威人物，而且知识非常渊
博；多次到过中国讲学，人更是非常友善，任何时候请教任何问题或是寻求帮助，从来都让你感到他不仅像一位学者．更是一位慈
祥的长者。本书的作者们基于欧美主要具有历史性的植物学研究机构，收集的文献可谓植物分类学史上从未有过。然而本书仍
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作者简介：Jinshuang Ma(马金双)，male，Ph．D．，Research Taxonomist，Brooklyn Botanic Garden，1000 Washington Avenue，Brooklyn，NY
11225～1099，USA (jinshuangma@bbg．org)。