全 文 ：园 　艺 　学 　报 　2007, 34 (1) : 71 - 74
Acta Horticulturae Sinica
Rece ived: 2006 - 06 - 11; Accepted: 2006 - 11 - 28
Supported by the National Natural Science Foundation of China ( 30571288, 30640032 ) , and by the M inistry of Education of China
( IRT0548, NCET20420734)3 E2mail: guoww@mail1hzau1edu1cn
Flower ing and Pollen Fertility of an Interspec if ic C itrus
Hexaplo id Soma tic Hybr id
GUO W en2wu3 , CHEN Chun2li, DENG Yu2yang, and DENG Xiu2xin
(N ational Key Laboratory of C rop Genetic Im provem ent, Huazhong A gricultural U niversity, W uhan 430070, Ch ina)
Abstract: Interspecific hexap loid somatic hybrid between J incheng orange and HR
(Ham lin orange + Rough lemon ) regenerated via electrofusion flowered seven years after
transp lantation in greenhouse. Flow cytometry analysis further confirmed its true hexap loid
nature. Its pollen stainability and germ inability was 4018% and 317% , respectively. In 2006,
the hexalpoid p lant flowered again and its pollen stainability was 2013%. Since there is no
natural citrus hexap loid p lant, this is the first report of fertile hexap loid somatic hybrid p lant in
C itrus.
Key words: C itrus; Hexap loid; Somatic hybrid; Flowering; Fertility evaluation
柑橘种间六倍体体细胞杂种开花及其花粉育性
郭文武 3 , 陈春丽 , 邓于洋 , 邓秀新
(华中农业大学作物遗传改良国家重点实验室 , 武汉 430070)
摘 　要 : 二倍体锦橙 +种间四倍体体细胞杂种 HR (哈姆林甜橙 + 粗柠檬 ) 的种间六倍体体细胞杂种
植株移入温室 7年后 (即 2004年 ) , 有 1株六倍体首次开花 , 花粉染色活力 4018% , 花粉萌发率 317% ;
2006年该植株再次开花 , 花粉染色活力为 2013%。由于自然界中没有柑橘六倍体 , 本研究是人工的方法创
造出可育的柑橘六倍体体细胞杂种植株的首例报道。
关键词 : 柑橘 ; 六倍体 ; 体细胞杂种 ; 开花 ; 育性评价
中图分类号 : S 666　　文献标识码 : A　　文章编号 : 05132353X (2007) 0120071204
C itrus cultivars are mainly dip loid with a few trip loid and autotetrap loid; polyp loid higher than tetrap loid
level, e. g. pentap loid, hexap loid and octop loid are rare (Barrett & Hutchison, 1981; Lee, 1988; O iyama &
Kobayashi, 1991). The well2established technique of p rotop last fusion and numerous allotetrap loids resulted
from this technique facilitated the fusion experiment between dip loid and tetrap loid in an effort to p rove
whether hexap loid p lant could be regenerated and if so, whether these hexap loid p lants are fertile.
Consequently, interspecific hexap loid p lants between dip loid J incheng and allotetrap loid somatic hybrid of HR
(Ham lin orange + Rough lemon) were p reviously p roduced ( Guo et al. , 2003 ). This hexap loid hybrid
p lant first flowered in 2004, and flowered again in 2006. Herein, it confirmed that the hexap loid p lant was
fertile. This is the first report of artificially created fertile hexap loid hybrid p lant in C itrus.
Acta Horticulturae Sinica Vol. 34
1　M a ter ia ls and M ethods
Materials: J incheng (C itrus sinensis‘J incheng’) + HR [ Ham lin orange (C. sinensis‘Ham lin’) +
Rough lemon ( C. jam bh iri) ] interspecific hexap loid somatic hybrid p lants were p roduced via electrofusion
( Guo et al. , 2003) , grafted onto Rough lemon rootstock, and transp lanted into greenhouse in 1998.
Ploidy analysis: Ploidy analysis was conducted in a Partec flow cytometry (D248161 Münster Germany).
App roximately 015 cm2 of young leaves were chopped in a p lastic Petri dish containing 014 mL Partec HR2A
buffer ( Partec high resolution nuclei extraction solution). After being filtered, the samp les were stained for 3
m inutes with 116 mL of HR2B buffer ( Partec high resolution DAP I staining solution) and the relative fluores2
cence of total DNA was measured. Each histogram was generated by analyzing at least 5 000 nuclei.
Pollen fertility assay: 1) Pollen grains: anthers of nearly opened flowers were harvested and pollens were
released and suspended with 4 drop s of Tween220 added to the final 25 mL ddH2 O. Pollen grains per anther
were calculated by haematocytometer with the following formula: pollen grains per anther = (pollen grains per
011μL ×10 ×1 000 ×4) /N (N refers to anther number). Pollen morphology and size: pollenswere brushed
on slide, and their diameters were measured by m icroruler with five repetitions, each repetition was with at
least 100 grains. 2 ) Pollen fertility: anthers were collected, and pollen stainability was determ ined using
015% acetocarm ine, by counting at least 1 000 grains. 3) Pollen germ inability was determ ined on agar medi2
um (018% agar + 16% sucrose + 10 - 6 mol/L boric acid) at 28℃ for 18 h by observing at least 1 000
grains.
2　Results and D iscussion s
One J incheng + HR hexap loid somatic hybrid p lant first flowered in 2004 after seven years of
transp lantation ( Fig11). The p loidy level of this p lant was p reviously determ ined by chromosome counting, to
further confirm its p loidy level, flow cytometry analysis using young leaf samp leswas conducted and the results
showed that the peak of dip loid J incheng control was around 100 ( Fig12) , that of the allotetrap loid HR was
200 ( Fig12) , while that of the J incheng + HR hybrid was 300 ( Fig12) , thus confirm ing its true and stable
hexap loid nature.
27
　No. 1 GUO W en2wu et al. : Flowering and Pollen Fertility of an Interspecific C itrus Hexap loid Somatic Hybrid　
　　Morphology observation of leaf and flower organ of J incheng + HR showed that its leaveswere thicker but
not so flat compared with its fusion parents ( Fig13, A ) , the flower size was bigger, the petal was thicker
( Fig13, B) , and the flower styles were aborted at different degrees ( Fig13, C).
F ig. 3　L eaf and flower m orphology com par ison of J incheng
orange + HR ( JHR) hexaplo id hybr id plan t
　　 Pollen fertility assay of this hexap loid hybrid
p lant in 2004 showed that its pollen stainability and
germ inability was 4018% and 317% , respectively.
This hybrid p lant flowered again in 2006 and its
pollen stainability was 2013% ( Table 1). Its pollen
fertility and pollen grains per anther in 2006 were
m uch lower than that in 2004 , possib ly due to the
Table 1　Pollen fertility of J incheng orange +
HR hexaplo id soma tic hybr id
Year
Pollen
stainability
( % )
Pollen
germ inability
( % )
Pollen
grains per
anther
Pollen
size
(μm)
2004 4018 317 16 970 4015
2006 2013 / 10 800 /
climate, tree nutrition condition and management level, since the growth of most potted citrus trees in 2006
was poor.
In recent years, there are increasing reports of hexap loid hybrid p roduction by p rotop last fusion in citrus.
For instance, fusions of tetrap loid Fortunella h indsii with Poncirus trifolia ta (M iranda et al. , 1997) , and
dip loid Amp lycarpa mandarin with tetrap loid somatic hybrid of sour orange + Flying dragon trifoliate orange
(Medina2U rrutia et al. , 2004) , both p roduced hexap loid hybrid p lants. Fusions between dip loids of Bonnaza
navel orange with C lausena lansium , Amp lycarpa mandarin with C235 citrange also regenerated hexap loid
hybrid p lants unexpectedly ( Guo & Deng, 1999; Medina2U rrutia et al. , 2004). But there is no report yet on
citrus hexap loid flowering. Since there is no natural hexap loid in citrus, this is the first report of artificially
created fertile citrus hexap loid hybrid p lant, which p rovided valuable high p loidy germp lasm for citrus p loidy
breeding and for p lant somatic cell genetic research.
37
Acta Horticulturae Sinica Vol. 34
References
Barrett H C, Hutchison D J. 1981. Occurrence of a spontaneous octop loid in apom ictic seedlings of a tetrap loid citrus hybrid. Proceedings of
　　 International Society of Citriculture, 1: 29 - 30.
Guo W W , Deng X X. 1999. Intertribal hexap loid hybrid p lants regeneration from electrofusion between dip loids of Citrus sinensis and its sexually in2
compatible relative, Clausena lansium. Theoretical and App lied Genetics, 98: 581 - 585.
Guo W W , Yu G H, Deng X X. 2003. Somatic hybridization between dip loids and an allotetrap loid somatic hybrid in Citrus. Proceedings of Interna2
tional Society of Citriculture, 145 - 146.
Lee L S. 1988. Citrus polyp loidy: origins and potential for cultivar imp rovement. Australian Journal of Agricultural Research, 39: 735 - 747.
Medina2U rrutia V, Madera K F L, Serrano P, Ananthakrishnan G, Grosser J W , Guo W W. 2004. New intergeneric somatic hybrids combining
Amblycarpa mandarin with six trifoliate / trifoliate hybrid selections for lime rootstock imp rovement. HortScience, 39: 355 - 360.
M iranda M, Motomura T, Ikeda F, Ohgawara T, Saito W , Endo T, Omura M, Moriguchi T. 1997. Somatic hybrids obtained by fusion between
Poncirus trifoliata (2x) and Fortunella h indsii (4x) p rotop lasts. Plant Cell Reports, 16: 401 - 405.
O iyama I, Kobayashi S. 1991. Citrus pentap loids from small seeds of dip loid ×dip loid crosses. HortScience, 26: 292 - 293.
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