全 文 ：文章编号 :100028551 (2008) 04242026
EFFECT OF ION IMPLANTATION ON B . mucilaginosus KNP414 AND SCREENING
FOR MUTANTS WITH HIGHER RELEASE OF PHOSPHATE AND POTASSIUM
HU Xiu2fang 　GAO Yuan2yuan 　FANG Qiong2lou 　WU Jin2guang 　CHEN Ji2shuang3
( Institute of Bioengineering , Zhejiang Sci2Tech University , Hangzhou , Zhejiang 310018)
Abstract :Effect of ion implantation on strain KNP414 of Bacillus mucilaginosus was investigated. Survival ratio of isolate
KNP414 was independent of the tested ions species and energy , but was highly dependent on their doses and the presence of
bacterial capsule. 14 phytate2degradation mutants were obtained from the spores implanted with N+ (20keV , 5 ×1015～5 ×
1016 ions cm - 2 ) , and their phytate2degradation capacities were 15 %～35 %. Amongst , 3 mutants ( KNP414204 , KNP4142
05 , KNP414212) with higher solubilizing capacities of mineral phosphate and potassium were identified. Their solubilizing
capacities of the two minerals increased by 1417 %～2715 % and 1612 %～2614 % , respectively. Mutant KNP414212 was
found to be able to dissolve 5713 % phytate in the optimized medium and the degradation ratio was comparatively stable in
seven continuous generations and during the storage process. In conclusion , ion implantation was an effective mutagenic source
for isolate KNP414 , since it increased the abilities to solubilize phosphate and potassium as well as to degradate phytate.
Meanwhile the screening method used in this study has been proved to be successful for rapid selection of phytate2degradation
mutants.
Key words : ion implantation ; Bacillus mucilaginosus ; mutant screening ; phytate degradation ; potassium and phosphate
solubilization
离子束注入技术选育胶质芽孢杆菌 KNP414
的解磷突变菌株
胡秀芳　高园园　方琼楼　吴金光　陈集双
(浙江理工大学生物工程研究所 ,浙江 杭州　310018)
摘 　要 :利用离子束注入对胶质芽孢杆菌 KNP414 进行诱变 ,获得可降解植酸的突变菌株。离子束注入
效应表明 ,菌株 KNP414 的存活率显著受离子束剂量及菌体荚膜的影响 ,但与所研究的离子种类及其能
量没有相关性。经离子束 N + (20keV , 5 ×1015～5 ×1016 ions cm - 2 ) 诱变后 ,筛选到 14 个植酸降解突变
株 , 它们对植酸的降解率为 15 %～35 %。其中的 3 个突变株 ( KNP414204 , KNP414205 , KNP414212) 分解
矿物磷和钾的能力也明显提高 ,分别增加 1417 %～2715 %和 1612 %～2614 %。在优化培养基中 ,突变菌
株 KNP414212 对植酸的降解率达到 5713 % ,且在连续培养及保藏过程中保持稳定。植酸降解突变株
KNP414212 的成功选育表明离子束为胶质芽孢杆菌的性状改良提供了有效途径。
关键词 :离子束注入 ;胶质芽孢杆菌 ;突变菌株筛选 ;植酸降解 ;磷和钾分解
收稿日期 :2007212201 　接受日期 :2008202229
基金项目 :国家高技术研究发展计划 (863 计划)项目 (2006AA10Z428)
作者简介 :胡秀芳 (19712) ,女 ,湖北黄石人 ,副研究员 ,从事资源微生物学研究。Tel :0571286843195 ,E2mail :huxiuf @zstu. edu. cn
通讯作者 :陈集双 (19622) ,男 ,湖南石门人 ,教授 ,从事微生物学研究。Tel :0571286843196 ,E2mail :chenjs @zstu. edu. cn　 　Both phosphate and potassium are major essential macronutrients for plant growth and development[1 ] .
024　 核 农 学 报　2008 ,22 (4) :420～425Journal of Nuclear Agricultural Sciences
However , they usually exist in the form of insoluble rocks ,
minerals , and organism. Phytate , due to its stability ,
constitutes 30 %～50 % of the total organic phosphate in
soil [2 ] , and increasingly high quantity of such kind of
phosphate has been accumulating in lands since organic
manure are used in agricultural practice. Presently , the
bioavailable phosphate and potassium are generally not
sufficient to meet the need of maximum crop growth , and
their availability has become a key limiting factor in crop
yields. Some microorganisms can solubilize inorganic
phosphate and potassium and degrade organic phosphate ,
thus invalid phosphate and potassium was released and to be
assimilated by crop [3 ] . Silicate bacteria have been found to
play a great role in the biotransformation of both phosphate
and potassium in soil[4 ,5 ] .
Silicate bacteria are rhizosphere microorganisms which
can decompose silicate salts , mineral in soil and release
soluble K, P , Si , Mn , etc. B . mucilaginosus , the typical
silicate bacteria with large capsule , were widely used in
metallurgic industry to leach impurities and in agriculture as
biofertilizer[6 ] . The use of B . mucilaginosus resulted in
significant increases in potato yields , without any adverse
effects[7 ] . All these functions of B . mucilaginosus are
based on its dissolution of inorganic materials. Since organic
phosphate , especially phytate , is a major source of
phosphate in soil , B . mucilaginosus should be endowed
with additional phytate degradation ability to provide more
free phosphate and thus not only to improve the growth and
yield of crops , but also to keep an amicable environment .
This paper will focus on strenthening the ability of B .
mucilaginosus KNP414 to release phosphate and potassium
in various pathways , especially by the degradation of phytate
through mutation. Genetic mutation induced by X2rays
irradiation , which has been used in breeding since 1927 , is
still an important method to improve the species of
organisms[8 ] . A variety of mutagenic source , such as
ultraviolet rays , X2rays , laser , neutron , and
chemomarphosis , have been successfully developed and used
in breeding in the following years. People are still attempting
to seek new mutagenic sources to improve mutational
spectrum and frequency in order to obtain efficient and
effective mutants.
Ion implantation , functioning well in semiconductor
and surface modification of materials , now evolved into a
new mutagenic method in breeding[9 ] . With the development
of space radiobiology and exobiology , biological effects of
ions were universally recognized[10 ] and the mutation induced
by ions has been widely proved in a wide range of
organisms[11 ,12 ] . Now , this kind of mutation , especially with
low energies , has become a new mutagenic origin and has
been widely used in crop and microbe breeding[13 ,14 ] .
Here , ion implantation has been used to induce the
mutation of isolate KNP414 and a method has also been
developed to screen the mutants with higher ability of
releasing phosphate and potassium.
1 　Materials and methods
111 　Cell culture and pretreatment
Strain AS1. 153 of B . mucilaginosus , which was a
common manufacturing bacterium for potassium biofertilizer
in China and was ordered from China General
Microbiological Culture Collection Center , was used as the
reference strain.
Isolate KNP414 of B . mucilaginosus was obtained from
soil in Zhejiang Province , China. After being activated in
slant Aleksandrov solid medium at 30 ℃ for 36h , a single
colony of isolate KNP414 was transferred to a shake flask
containing Aleksandrov liquid medium and the liquid revised
medium (R2Aleksandrov medium) and cultured at 30 ℃for
the formation of spores with and without capsule ,
respectively. Spores were collected and prepared into a
suspension of 105 cells ml - 1 with saline water. 015ml of the
cell suspension were spread on aseptic glass and air2dried at
room temperature. Samples were prepared in the same way
as above to test desiccation effect on the survival ratio.
Aleksandrov medium contained 015 % glucose , 012 %
Na2 HPO4 , 0105 % MgSO4 ·7H2O , 010005 % FeCl3 ,
0101 % CaCO3 and 011 % mineral . R2Aleksandrov medium
comprised 1 % starch , 012 % yeast extract , 0105 % MgSO4
·7H2O , 010005 % FeCl3 and 0115 % CaCO3 .
112 　Ion implantation and survival analysis
Spores of isolate KNP414 were implanted with nitrogen
or argon ions using an IM2200 ion implanter ( Institute of
Biophysics , Chinese Academy of Sciences) . The doses of
ions for implantation ranged from 1 ×1014 to 1 ×1016 ions
cm
- 2
, and the energy from 10 to 30keV. Control samples
were placed in target chamber but without ion implantation to
124　4 期 离子束注入技术选育胶质芽孢杆菌 KNP414 的解磷突变菌株
test the vacuum effect . The treatments of all the samples
were performed at room temperature.
Cell survival rate was determined by the conventional
colony2forming assay. After being implanted , spores were
washed out from glass plates and were incubated in 10 ml of
saline water at room temperature for 30 min with gentle
agitation , and then they were diluted into a series of
concentrations. Dilutions were plated on Aleksandrov plates
and incubated at 30 ℃for 3～5d for recording the number of
colonies. Three plates were used for each dilution , and each
experiment was performed in triplicate.
113 　Mutant screening
Ions2implanted spores were washed out with liquid
phytate screening medium (PSM) and incubated at 30 ℃for
8～ 12h to allow the germination of spores. The PSM
contained 015 % calcium phytate , 110 % glucose , 0105 %
KCl , 0105 % MgSO4 ·7H2O , 0101 % MnSO4 ·H2O and
01001 % FeSO4 ·7H2O in distilled water. The germinated
bacteria were properly diluted into the liquid PSM and they
were distributed in the form of 12ml aliquots into sterilized
482well plates until each well contained 1 to 3 cells. The
parent bacteria and uninoculated medium were treated in the
same way as the controls. The 482well plates were incubated
at 30 ℃ for 7d and were centrifuged at 14000rΠmin for
20min. 100μl of supernate was transferred into 962well
quartz plates , and molybdovanadate reagent[15 ] was added
into each well . The plate was shaken slightly to obtain the
homogeneous mixture and the optical density (OD) values at
700 nm were measured 30 min later to determine the content
of available phosphate. The cultures with at least 15 %
higher available P than controls were purified on PSM plates
as the mutants.
The mutants were further selected on the Aleksandrov
medium containing bromophenol blue. Colonies with clear
zones were selected as the aimed mutants and were kept for
the following experiments.
114 　Phytate degradation
The mutants were inoculated in liquid PSM and
incubated on a shaker (200rΠmin) at 30 ℃ for 24h. They
were re2inoculated into the shake2flask containing liquid
PSM with sodium phytate instead of calcium phytate and
incubated at 30 ℃for 7d. The cultures were centrifuged at
10000rΠmin for 10 min and the supernatant was used for
analysis of phytate residue as describe by Nickel et al[16 ] .
A 017 ×1510cm column was clamped vertically and
packed with 015g anion exchange resin (AG12X8) , and was
washed with deionized water. 1 ml of the supernatant was
added to the column. The column was washed with 15 ml of
011 molΠL NaCl. The phytate was eluted with 15 ml of 017
molΠL NaCl and the elution was collected in a 100 ml micro2
Kjeldahl flask. The elution was digested with concentrated
H2 SO4 (015 ml) and HNO3 (310 ml) and solubilized with 15
ml of deionized water. The solution was made to a volume of
50 ml with deionized water. The content of phosphate was
detected utilizing the molybdo2vanado2method and that of
phytate was calculated by the principle that one molecule of
phytate contained six molecules of phosphate[17 ] .
115 　Dissolution of minerals
The abilities of the mutants to solubilize the minerals
were tested in the liquid Aleksandrov medium with
phosphorite (assigned as GYfrom Guiyang Province , China)
and potassium minerals (potassium feldspar) as the only P
and K source , respectively. Strain AS1. 153 was used as the
control .
Quantitative estimation of phosphate and potassium
solubilization was carried out in Erlenmeyer flasks containing
20 ml of Aleksandrov liquid medium. The flasks were
incubated at 30 ℃for 7 d on a shaker with 200 rΠmin. The
cultures were diluted 1∶1 (V∶V) using 011 molΠL HCl , and
bacterial cells were harvested after centrifugation at 12000rΠ
min for 10 min. The contents of phosphate and potassium
solubilized in the supernatants were tested utilizing molybdo2
vanado2method and atomic absorption spectrometry[14 ] .
Values are given as means ±S. D. for each sample , and
differences were considered to be significant at the P < 0105
level .
116 　Analysis of genetic stability
The genetic stability of mutant KNP414212 was
analyzed in the optimized PSM (OPSM) which contained
1 % glucose , 0105 % (NH4 ) 2 SO4 , 015 % calcium phytate ,
015 % MgCl2 , 0103 % MgSO4 ·7H2O , 0105 % KCl ,
0101 % MnSO4·H2O and 01001 % FeSO4·7H2O , pH 710.
On OPSM , the mutant was transferred 7 times on end , and
each generation was cultured to test the abilities of phytate2
degradation. The capacity was also detected in a 22month
interval within 22year2long storage at 4 ℃.
224 核　农　学　报 22 卷
2 　Results and discussion
211 　Survival ratio of bacteria under ion implantation
The survival of bacteria is a prerequisite for screening
ion2induced mutants. Isolate KNP414 is a kind of spore2
forming silicate bacterium with large capsule. In
Aleksandrov medium , it produced large capsule , but no
capsule was formed in the revised medium ( Fig11) . The
effect of capsule on ion implantation was further investigated
( Fig12 ) . Ion implantation significantly decreased the
survival rate of isolate KNP414 as the dose increased.
Spores with capsule presented higher survival ratio than those
without capsule under the same condition. Experiments
indicated that desiccation and vacuum exerted little effect on
the survival ratio ( data not presented) . Therefore , spores
without capsule were used in the following experiments.
Fig. 1 　Spores morphology of isolate KNP414 with (A)
and without capsule (B) cultured in different medium
图 1 　不同培养基中菌株 KNP414 的芽孢形态
(A :有荚膜 ;B :无荚膜)
Fig. 2 　Survival ratio of isolate KNP414
implanted with N + ions
图 2 　N + 离子注入对菌株 KNP414 存活率的影响
Effect of energy and species of ions on the survival of
spores without capsule was investigated and presented in
Fig13. With the increment of energy , implantation of N +
induced higher lethal ratio of isolate KNP414 , but no
significant difference was observed between the treatments of
various N + energies. Under the condition of the same energy
and dose , implantation of Ar + and N + resulted in similar
survival rates. When the ion dose increased to higher than 5
×1015 ions cm - 2 , survival rate of the spores was reduced to
less than 5 % , but enough spores were still alive for mutant
selection. It is reported that there exists a“mutagenic belt”
where the density of ion is not high enough to kill the cell ,
but enough to induce mutations[18 ] , and that the dose of ion
implantation inducing more than 90 % lethality is optimal to
obtain mutants. Therefore , at least 5 ×1015 ions cm - 2 was
used as the doses to induce mutation in the followings.
Fig. 3 　Survival ratio of isolate KNP414
implanted with various ions
图 3 　不同离子注入对菌株 KNP414 存活率的影响
It is obvious that dose of ions markedly influenced the
survival of isolate KNP414 , but species and energy used in
this study did not have significant effect . Our result was
consistent with the conclusion from other research[19 ,20 ] .
212 　Isolation of mutants
Based on the dose2dependent effect , spores without
capsule were implanted with N + (20keV , 5 ×1015～5 ×1016
ions cm - 2 ) to obtain mutants. After culturing in R2
Aleksandrov medium for 48 h , spores of isolate KNP414
were pretreated , implanted with N + ions , and were cultured
in liquid PSM for mutant screening.
Compared with controls , the cultures with the
supernatant containing 15 % or higher available P were
collected from the 482well plate and purified as the mutants.
In the implantation treatment with 5 ×1015 ionsΠcm2 , 5
satisfying mutants were collected , and the mutation ratio was
calculated to be 015 %. In the treatment with 1 ×1016 ions
cm
- 2
, 9 mutants were obtained and the mutation ratio was
112 % , even though the total amount of spores that survived
was greatly reduced. The mutants were named as KNP4142
324　4 期 离子束注入技术选育胶质芽孢杆菌 KNP414 的解磷突变菌株
01 , KNP414202 , KNP414203 , until KNP414214.
Purified clones of mutants were transfered into 100 ml
of liquid PSM. The culture flask was incubated at 30 ℃on
an orbital shaker at 200 rΠmin for 7 d. Supernatants were
harvested after centrifugation at 10000 rΠmin for 10 min , and
were used to estimate the content of phytate residue. The
phytate degradation ratios of the selected mutants ranged
from 15 % to 35 % in the liquid medium.
All the 14 mutants were re2selected on the Aleksandrov
medium with the presence of bromophenol blue. Three of the
mutants , KNP414204 , KNP414205 and KNP414212 , formed
clear zones steadily , and they were kept for the following
experiments.
213 　Dissolution of minerals
The capacities of the three mutants to solubilize
phosphate and potassium minerals were investigated in the
liquid Aleksandrov medium with the phosphorite and
potassium minerals as the only P and K source ,
respectively. The mutants showed significantly higher release
ability of phosphate minerals GY than the parent strain did.
Compared with the parent strain , the solubilizing capacities
of mutants KNP414204 , KNP414205 and KNP414212
increased by 1516 ±0189 % , 1417 ±0177 % and 2715
±1138 % , respectively. The similar results were obtained
in the test of potassium mineral solubilization. In the
medium with potassium feldspar , the increment value of
solubilization capacities of the mutants KNP414204 ,
KNP414205 and KNP414212 were 1915 ± 0197 % , 1612
± 0185 % and 2614 ± 1132 % , respectively. Mutant
KNP414212 was confirmed to have the highest capacities to
solubilize both GY and potassium feldspar in the repeated
experiments.
Improvement of availability of phosphate and potassium
is a pivotal way to promote the growth of crops. The three
mutants might be the potential inoculant for production of
microfertilizers , since they had the increased capacity of
phytate degradation as well as higher potassium and
phosphate solubilization ability.
214 　Confirmation of genetic stability
The PSM medium was optimized as OPSM to reach the
highest potency of phytate degradation. In the liquid OPSM ,
the parent strain and the mutant KNP414212 were cultured to
detect the ability of phytate degradation ( Fig14) . Mutant
KNP414212 showed much higher capacity in OPSM than in
PSM. Cultured in the same conditions , the phytate
degradation rate of mutant KNP414212 in OPSM was 5713
±1195 % while phytate degradation rate of isolate KNP414
was less than 6 ± 0115 %. Mutants KNP414204 and
KNP414205 also had higher phytate2degradation abilities
(3718 ù 1156 % and 2912 ù 1122 % , respectively) in
OPSM.
Fig. 4 　Phytate degradation of mutant KNP414212
and isolate KNP414 in PSM and OPSM medium
(33 P < 011 phytate degradatio of mutant
KNP414212 in OPSM versus that in PSM)
图 4 　突变菌株 KNP414212 与出发菌株 KNP414
在不同培养基中的植酸降解作用
Genetic stability was the critical factor for mutant to
retain its properties. The phytate degradation stability of
mutant KNP414212 was tested in OPSM (Fig15) . Capacities
of phytate degradation were similar in seven continuous
generations , and the same results were obtained in 22month
interval in the period of 22year2long storage ( data no
presented) . So the mutant KNP414212 was relatively stable
in terms of phytate degradation and can be used in practice.
Fig. 5 　Stabilities of phytate degradation of
mutant KNP414212 in different generations
图 5 　突变菌株 KNP414212 与传代过程中
植酸降解能力的稳定性
3 　Conclusion
In conclusion , 14 mutants were obtained utilizing
424 核　农　学　报 22 卷
methods of ion implantation and high2throughput screening.
Compared with the parent strain , three of the mutants were
analyzed to have much higher phosphate and potassium
dissolution , 1417 %～2715 % increments value in mineral
solubilization and 2712 %～5713 % in phytate degradation.
The results indicated that ion implantation was an effective
mutagenic source , and the screening method was proved to
be successful for rapid selection of phytate degradation
mutants.
Acknowledgements 　Ion implantation was performed
at Institute of Biophysics , Chinese Academy of Sciences.
We are thankful to Professor Yu Zengliang and Dr. Yu Long
for technical support .
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