全 文 ：西北植物学报 , 2008 , 28(4):0697-0703
Acta Bot.Boreal.-Occident.Sin.
文章编号:1000-4025(2008)04-0697-07 ＊
根瘤形成过程中豆科植物根毛
和根外层传递细胞的诱发
林树燕1 ,吴均章2 ,韩素芬1＊
(1南京林业大学 ,南京 210037;2 苏州大学 ,江苏苏州 215000)
摘 要:对 7 种豆科植物接种根瘤菌后根部的形态和内部结构进行了研究.结果表明:根瘤菌可诱发根瘤形成部位
根段的根毛增生 、形变和根外层传递细胞的发育.根外层传递细胞发生在根毛伸长形变时期 , 一直可持续到根瘤形
成 ,传递细胞壁内突发育过程是先由根表皮细胞外切向壁一侧细胞质膜向细胞质内陷形成囊状壁傍体 , 次生细胞
壁物质在初生壁上沉积并逐渐充满囊状体 ,最终形成传递细胞典型的壁内突结构.根瘤形成过程中根外层传递细
胞的诱发与培养方式(水培 、固培)没有直接关系.在不接菌的对照苗的根段内未发现壁内突结构 ,研究证明豆科植
物根外层传递细胞的形成是由根瘤菌诱导所致.
关键词:豆科植物;根瘤;根瘤菌;根毛;传递细胞
中图分类号:Q948.12 +2.2 文献标识码:A
Inducement of Root Hair and the Root Epidermal Transfer Cells
during the Formation of Root Nodule in Leguminous Plants
LIN Shu-yan1 ,WU Jun-zhang2 ,HAN S u-fen1＊
(1 Nanjing Forest ry Universi ty , Nanjing 210037 , China;2 Suzh ou Universi ty , Suzhou , Jiangsu 215000 , China)
Abstract:The mo rpho logy and anatomy of 7 legume species wi th the rhizobia w ere investig ated.The re sults
show ed that the hyperplasy o f root hai r , roo t deformation and the development of the roo t epidermal t rans-
fer cells we re induced by rhizobia.The transfer cells began to develop f rom the phase of ro ot hair defo rma-
tion , and they could live until the fo rmation o f the nodule.Cell membrane ingrow ths were f irst observed at
the initial stage of the roo t hai r deformat ion , then the w all material g radually depo sited and finally t ransfer
cells formed.T he development of transfer cells had no relation wi th the medium whether i t w as solid o r liq-
uid.The transfer cell did no t exist in the ro ot w hich w as no t inoculated by the rhizobia.
Key words:Leguminous plants;ro ot nodule;rhizobia;roo t hair;transfer cell s
In 1968 , Gunning[ 1 ,2] named the special phloem
parenchyma cells as t ransfer cells.Now the cell with
wall ing row ths are all called t ransfer cells
[ 3-5] , which
mainly serve as a structure specialized in relation to
short distance t ransport of solutes.The cell w as widely
distributed in the different organs of the different spe-
cies , especially in the vascular system such as stems ,
nodes , leaves and reproductive organs[ 6] .It had been
repo rted there were transfer cells near to the vascular
bundle sy stem of the root nodule in some legume
plants such as pea , kidney bean , broad bean , the soy-
bean and a species of cowpea.
＊ 收稿日期:2007-08-06;修改稿收到日期:2008-03-31
基金项目:国家自然科学基金(39970601)
作者简介:林树燕(1976-),女(汉族),博士生 ,讲师 ,主要从事木本植物根瘤方面的研究.
＊通讯作者:韩素芬 ,女 ,教授 ,博士生导师.E-m ail:l rx@njfu.com.cn
Moreover , the cells of phloem in soybean had wall
ing row ths.It w as also reported that the epidermal cells
near to the roo t tip could develop the transfer cells
when sunflower g rew in the aqueous solution without
i ron
[ 7] .However , the research about t ransfer cells in
plant root was rare.In 1996 , Han et al.[ 8] observed
root epidermal transfer cells of locust seedling af ter in-
oculating the rhizobia by TEM.The inducement of
transfer cells during the formation of Robinia pseud-
oacacias root nodule was reported in 2002[ 9] .In 2004
and 2005 ,Wu Junzhang[ 10 , 11] observed the t ransfer cells
in the epidermis of roots of Astragalus sinicus and Al-
bizzia julibrissin af ter inoculation.In this study , we
observed the deformation of root hair and the induce-
ment of the root epidermal and exodermis t ransfer cells
in 7 legume species.The relationship between the de-
velopment of the root epidermal t ransfer cells and the
formation of the root nodule was studied.
1 Materials and methods
1.1 Materials
Seeds:locust (Robinia pseudoacacia), silk tree
(Albizzia julibrissin), peashrub(Caragana sp.), clo-
ver (Tri f olium sp.), Chinese milkvetch (Astragalus
sinicus), alfalfa (Medicago sativa), soybean (Glycine
max).
Rhizobia:rhizobia of locust 87-1-1 , silk tree 2-18
and peashrub 94-161 were derived from the roo t nod-
ules respectively;rhizobia of clover ,Chinese milkvetch ,
alfalfa and soybean were obtained from Nanjing Agri-
cultural University.
Medium:①YEM:manni tol 10 g , K 2HPO4 0.5
g ,MgSO 4 7H 2O 0.2 g , NaCl 0.2 g , yeast ex tract
0.8 g , CaCO 3 5 g , 0.5% NaM oO4 4 mL , 0.5%
H 3BO 3 4 mL ,H 2O 1 000 mL.
②NF:CaCl2 2H 2O 1 329 mg , KH 2PO 4 100
mg , N a2HPO 4 150 mg , MgSO 4 7H 2O 120 mg ,
FeC4H 5O 7 5 mg , H3BO 3 6.2 mg , MnSO4 4H 2O 0.
17 mg , ZnSO 4 7H 2O 0.28 mg , CoCl 6H2O 0.24
mg , CaSO 4 5H2O 0.025 mg , NaM oO4 2H 2O 0.
024 mg ,pH 6.5.
1.2 Methods
1.2.1 The cultivation and inoculation of aseptic seed-
l ings The seeds mentioned above w ere t reated 30 s
with 70% alcohol , sterilized 7 ～ 10 min with 0.1%
Mercuric chloride ,washed 4 times with aseptic water ,
then soaked in aseptic water.After these seeds were
swelling , they were moved to tubes with filter paper to
cultivate under 30℃.When the roo t w as 4 ～ 5 cm
long , the seedling s were inoculated w ith the corre-
sponding rhizobia for 2 h.Then the seedlings were
t ransmitted to the bot tle with NF aqueous solution or
the culture dish with NF agar-solid medium.The cul-
ture room had an illumination of 2 000 lx for 14 h ev-
ery day with temperature 25℃.At the same time , we
took the aseptic seedlings as a control.During the cul-
tivation ,watch a series of changes of the roots and take
photos.
1.2.2 Preparation for light microscopy The root
segment with root hair deformation and the same part
of aseptic seedlings were fixed 48 h wi th 50% FAA ,
washed 3 times with 50% alcohol ,2 h each time , then
dehydrated in alcohol series(70%,80%,95%, 100%,
100%,2 h each time).After dehydration root segments
were put in Technovit 7 100 to infiltrate for 24 h , then
put in the new-made rapid solidification on buried
board , finally made the semi thin sections with 5 μm ,
which were stained with PAS (periodic acid and
Schiffs reagent), dried naturally , dehydrated in pure
alcohol 2 times ,2 min each time ,needed to be t ranspar-
ent with xylol for 2 min and sealed by the neutral glue ,
then these slices were examined and photog raphed.
1.2.3 Preparation for TEM The corresponding root
segments were fixed in 3% ～ 4% glutaraldehyde
(componded with neutral PBS);The deforming root
segment of the locust seedling during dif ferent period
(root hair deformation , a little expanding , small nod-
ule ,big nodule)and the non-inoculated root segment
were all fixed with 3% glutaraldehyde , then postfixed
in aqueous osmium tetroxide after w ashing with w ater
2 times , dehydrated by the alcohol series , infiltrated
them in the resin Epon 812.At last the ult rathin sec-
tions w ere cut by LKB-V type machine , stained with u-
ranyl acetate for 15 min and lead citrate for 20 min.
2 Results and analysis
2.1 The root morphological changes
After inoculating with the co rresponding rhizobia ,
698 西 北 植 物 学 报 28 卷
the number of roo t hair of 7 legume species increased
obviously , and the root hair showed obvious deforma-
tion especially the part where the root nodule would
form.But there were some dif ference among the spe-
cies.The seedling s inoculated with rhizobia not only
produced much deformed root hairs , but also fo rmed
root nodules in these deformed parts(PlateⅠ,1),while
the control seedlings of silk hardly see root hairs(Plate
Ⅰ,2).The root hairs of the locust seedling inoculated
with rhizobia had some kinds of deformed shapes.The
top of root hairs were expanding or had the irregular
shape or took the shape of calabash firstly(PlateⅠ,3).
Then they extended crookedly to the shape of hook or
the fo rm of bransh or the wave shapes(PlateⅠ,4).Af-
terwards the root hairs of the root segments growed in-
tensively thick (Plate Ⅰ, 5)and they may survive no
less than 10 day s.The part of the deformed root seg-
ments gradually formed the root nodules finally (Plate
Ⅰ,6 ～ 8).While the root hai rs of the control seedlings
and the roo t segments where there w erent nodules af-
ter inoculating with rhizobia grew normally (Plate Ⅰ,
9), and they only survived 4 to 5 days.For clover ,
peashrub , alfalfa , Chinese milkvetch and soybean , their
root hairs took the crooked shape ofZ, the top of the
root hair had some shapes , such as the ducks head , the
expanding and crooked branch af ter inoculating with
rhizobia(Plate Ⅰ,10 ～ 12).But the roots of the control
seedlings w ere normal and no deformation (Plate Ⅰ,13
～ 15).The results indicated that the inducement of
rhizobia resulted in the intensive thickness of root
hairs , the deformation of the root hairs and the forma-
tion of root nodules.
2.2 The position and the condition of root epider-
mal transfer cells in leguminous plants
By examing lo ts of semi-thin and ultrathin sec-
tions ,we observed many transfer cells ,which distribu-
ted in epidermal cells , exodermis cells and even in the
root hair cells.The quantities and position of t ransfer
cells varied in dif ferent species.The transfer cells of
Chinese milkvetch were in the epidermal cells and the
root hai r cells (Plate Ⅱ, 1).To locust seedling , they
were mostly in epidermal cells and the root hair cells ,
few in exodermis (Plate Ⅱ, 2).For clover , alfalfa ,
peashrub , silk t ree and soybean , the transfer cells dis-
t ributed in epidermal cells and exodermis(Plate Ⅱ,3 ～
7).But to these 7 species of legume without rhizobia
inoculating , there was no w all ingrowths in epidermis ,
exodermis and root hair cells(Plate Ⅱ, 8 ～ 14).There
w as also no t ransfer cells in the root inoculated with
rhizobia but no deformation(PlateⅡ,15).While to the
aseptic seedling of locust cultured in the medium with-
out i ron , there w ere few transfer cells in the root epi-
dermal cells ,but i t w as very rare(PlateⅡ,16).
There were some transfer cells in the position
w here the root nodule w ould form whether the seed-
ling s grew in the liquid or solid medium.So it can be
concluded that the transfer cells w ere induced by the
rhizobia during the formation of root nodules.
2.3 The development of the transfer cells of locusts
root segments
A series of morphological changes happened in the
root of locust seedling inoculated with rhizobia.Some
transfer cells in different period were examined from
the ult ra-thin sections which were made with the de-
formed root segments during the different development
periods(root hair defo rmation , the dense root hai rs and
a lit tle expanded root segments , small nodules , big nod-
ules).At the phrase of root hair deformation , transfer
cells were observed in the root epidermis(Plate Ⅲ,1);
When root hair was thick and root was a little expan-
ding , a lot of transfer cells w ere in root epidermal and
exodermis cells ,even in some root hair cells(Plate Ⅲ ,
2 , 3);When the expanding parts became small nod-
ules , root epidermal cells and exodermis cells were
mostly the t ransfer cells and lots of continuous transfer
cells w ere at the opposite side of root nodule(Plate Ⅲ ,
4).At last , there were still few disinteg rated transfer
cells near the nodule (Plate Ⅲ, 5).The obvious w all
ingrowths were the typical character of transfer cells in
w hich the ly toplasm w as very thick with a big and
round nucleus.
The transfer cells firstly grew in the extangential
w all of epidermal cells w hen the root hair of locust was
deformed.The number of t ransfer cells increased with
the development of the root nodule , and they existed
until the nodule formation.Transfer cells dist ributed in
the root epidermal cells or the exodermis cells or the
root hair cells.The formation of wall ingrowths was
6994 期 林树燕 ,等:根瘤形成过程中豆科植物根毛和根外层传递细胞的诱发(英文)
that the cell membrane fi rst ly grew inw ard to form a
space shaped like a nipple (Plate Ⅲ, 6), then the wall
material gradually deposited (Plate Ⅲ ,7 , 8), finally it
developed wall ing row ths(Plate Ⅲ,9).
3 Discussions
The dense deformed root hairs and the t ransfer
cells were all observed in the deformed root segments
of all 7 legume species after inoculating wi th rhizobia ,
while for the control seedlings , the root hairs were nor-
mal and there were no transfer cells.So it was sure that
the root morphological changes w as direct ly related
with the formation of nodule , and the transfer cells
were induced by rhizobia.
During the fo rmation of locust roo t nodule , the
epidermal transfer cells developed fi rst ly in the period
of root hair deformation , and they lived until the for-
mation of root nodule.The development of the w all in-
g row ths were as follow s:at first the cell membrane
grew inw ard , then the w all ingrowths w ere gradually
filled wi th the wall material.It was different to former
explanation that the ing row ths of secondary wall de-
veloped firstly and then the ingrowths of cell mem-
brane[ 7] .
During the formation of nodule , there are some
morphological changes such as the thickning and defor-
ming root hairs and the survival time of roo t hairs and
the development of transfer cells.These changes not
only benefited the invading of the rhizobia but also in-
creased the absorption and transport of water and all
kinds of nutritious materials.During the formation of
nodule ,it needs much more w ater and other nutritious
material.So the morphological changes fi t wi th the re-
quirement of nut rition , which demonst rated the consis-
tency between morphological changes and physiological
function
[ 5] .
The paper studied the development of transfer
cells during the formation of nodule , which offered a
base to give a further research on the cellular localiza-
tion and the function of transfer cells.
References:
[ 1] G UNNING B E S , PATE J S.T ran sfer cell s———plant cell s w ith w all ingrow ths , specialized in relation to sho rt di stan ce t ran sport of solu-
t ions-thei r occurrence , st ructu re , and developm ent[ J] .Protop lasma , 1969 , 68:107-133.
[ 2] G UNNING B E S , PAT E J S.Cell s w ith ingrow ths(t ransfer cells)in the placenta of ferns[ J] .P lan ta , 1969 , 87:271-274.
[ 3] PA TE J S.Vascular t ran sfer cel ls in angiosperm leaves a taxonomic and morphological su rvey[ J] .Protop lasma , 1969 , 68:135-156.
[ 4] FOLSOM M W.Change in t ransfer cell dist ribu tion in the ovule of soyb ean af ter fert ilizat ion[ J] .Can.J .Bot., 1986 , 64:965-972.
[ 5] 卡特 E G.植物解剖学:细胞与组织[ M] .北京:科学出版社 , 1986:184-190.
[ 6] G UNNING B E S.T ransfer cells and their roles in t ransport of solut ions in plants[ J] .S ci.Prog.O x f ., 1977 , 64:539-568.
[ 7] 李正理 ,张新英.植物解剖学[ M] .北京:高等教育出版社 , 1984:76-98.
[ 8] HAN S F(韩素芬), GAN X H(甘习华), HUANG J SH(黄金生).The ch anges of the root epidermis m orphology and ult ra-s t ructure of lo-
cu st af ter inoculat ing rhizobia[ J] .S cience o f Forestr y(林业科学), 1998 , 34(4):109-110(in Chinese).
[ 9] LIN SH Y(林树燕), GAN X H(甘习华), HUANG J SH(黄金生), et al.Inducement of t ransfer cell s an d u lt rast ructural observat ion of
root parts du ring the formation of Robinia pseudoacacias root n odu le[ J] .Journal o f Chinese Electron Microscop y Society(电子显微学
报), 2002 , 21(2):134-137(in Chinese).
[ 10] WU J ZH(吴均章), HAN S F(韩素芬), GAN X H(甘习华).Observat ion on th e t ransfer cel ls in the epiderm of roots of Astrag alus sini-
cus af ter inoculat ion by rhizobia[ J] .Journal o f Nanj ing Forestr y Univer sity(南京林业大学学报), 2004 , 28(1):81-83(in Chinese).
[ 11] WU J ZH(吴均章),GAN X H(甘习华), HAN S F(韩素芬).Proli feration of root hai rs and formation of t ran sfer cells of root layers in
A lbiz z ia ju libr issin induced by rhizobia[ J] .S cience o f Forest ry(林业科学), 2005 , 41(6):179-181(in Chinese).
700 西 北 植 物 学 报 28 卷
Plate Ⅰ The root morphological changes of 5 legume species
Fig.1.The deform ed root hairs of silk seedlin g after inocu lating wi th rhizobia , ×50;Fig.2.The root of asep tic silk s eedling , almost no root
hai rs , ×20;Fig.3.The deformed root hairs of locu st seedlings af ter inocu lating w i th rhizobia , ×50;Fig.4.The SEM micrograph of deformed
root hai rs , ×150;Fig.5.Th e den se deformed root hai rs of locust seedling , ×20;Fig.6.T he root nodule of locus t , ×20;Fig.7.T he SEM micro-
g raph of the nodule , ×90;Fig.8.The root nodule of locus t , ×20;Fig.9.T he normal root hairs of aseptic locu st seedlings.there w as n o deforma-
t ion , ×20;Fig.10.T he root of aseptic clover seedling , almost n o deformed root hairs , ×20;Fig.11.The deformed root hairs of alfalfa seedling
af ter inoculat ing wi th rhizobia , ×50;Fig.12.The deform ed root hai rs of Chines e milkvetch seedling after inoculat ing wi th rhizobia , ×50;Fig.
13.The deform ed root hairs of clover seedling af ter inocu lat ing w ith rhizobia , ×50;Fig.14.The root of asept ic alfal fa seedlin g , almost no de-
f ormed root hai rs , ×20;Fig.15.The root of asep tic Chinese milkvetch seedling , almos t no deformed root h ai rs , ×20.
7014 期 林树燕 ,等:根瘤形成过程中豆科植物根毛和根外层传递细胞的诱发(英文)
Plate Ⅱ The root cross sect ions of 7 leguminous plants
Fig.1.The part root cros s section of C hinese milkvetch seedling af ter inoculating w ith rhizobia , the root epidermal cell developed th e t rans-
f er cell s , ×500;Fig.2.The part root cross s ection of locus t seedling af ter inoculating w ith rhizobia , the hai r cells developed the t rans fer cell s , ×
500;Fig.3.The part root cross section of clover seedling after inocu lating wi th rhiz obia.the t ransfer cells developed in the epidermal cel ls and in
th e exodermis , ×500;Fig.4.T he part root cross s ection of alfalfa seedling af ter inoculat ing w ith rhizobia.Th e w all ingrow th s in th e root epider-
m al cell and exodermis , ×500;Fig.5.Th e part root cross sect ion of peashrub seedlin g after inocu lating wi th rhizobia.The w all ingrow th s in the
root epidermal cells an d exodermis , ×300;Fig.6.The part root cros s section of silk s eedling af ter inoculat ing w i th rhiz obia.The root epidermal
cel ls and exodermis developed th e t ran sfer cel ls , ×500;Fig.7.T he TEM micrograph of s oyb ean root epidermal t ran sfer cells af ter inoculating
w i th rhiz obia.The w all ingrow ths in the epidermal cell , ×10 000;Fig.8.The part root cross sect ion of asept ic Chinese milkvetch seedling , no
t ransfer cel l , ×500;Fig.9.The part root cross s ection of asept ic locust seedling , no t ransfer cell , ×500;Fig.10.T he part root cross section of a-
s ept ic clover seedling , no t ran sfer cel l , ×500;Fig.11.The part root cros s section of aseptic alfalfa seedling , no t ransfer cell , ×500;Fig.12.The
part root cross sect ion of asept ic peash ru b seedling , no t ran sfer cell , ×300;Fig.13.T he part root cros s sect ion of asept ic silk seedling , no transfer
cel l , ×300;Fig.14.The part root cross sect ion of asept ic s oybean seedling by light microscope , no t ransfer cell , ×300;Fig.15.T he epidermal
normal cel ls of normal root segm ents af ter inocu lat ing wi th rhiz obia , ×5 000;Fig.16.The few epidermal t ransfer cells of asept ic seedling w hich
g row s in the NF cul tu re solut ion wi thout iron , ×6 000.
702 西 北 植 物 学 报 28 卷
Plate Ⅲ The T EM micrograph of th e root epiderm al cells of locust seedlings grow ing in the di fferent ph rases and the development of
t ransfer cell s
Fig.1.The root epidermal t rans fer cell s of root segments w ith deformed root hai rs af ter inoculatin g wi th rhizobia , ×8 000;Fig.2.The con-
t inu ou s root epidermal t ransfer cells during the period of the dense root hai rs , ×2 000;Fig.3.T he t ran sfer cel l in root hai r cell , ×5 000;Fig.4.
The root epidermal t ran sfer cells w hen the root segment is a lit t le expanding , ×5 000;Fig.5.T he root remnant t ransfer cel ls on the opposi te side
of the big n odu le , ×15 000;Fig.6.Membrana ingrow th s on the init ial s tage of the fo rm at ion of the t ransfer cells , ×8 000;Fig.7 , 8.The wall ma-
t erial s are deposited , ×10 000;×15 000;Fig.9.Wall ing row ths on ex tangential w all of the form ed t ran sfer cel ls , ×8 000.
7034 期 林树燕 ,等:根瘤形成过程中豆科植物根毛和根外层传递细胞的诱发(英文)