Page 6 - Molecular Plant Breeding

Molecular Plant Breeding 2011, Vol.2, No.17, 119-122
http://mpb.sophiapublisher.com
120
methods which began in the 1980 s have been relied
on for the isolation of egg cell from maize (Kranz and
Lorz, 1993), wheat (Kovacs et al., 1995), Rice (Zhao
et al., 2000). Corn is by far the only case that achieved
in vitro fertilization using isolated egg cell obtained
from enzyme dissection method. Another method,
micro-dissection was developed in the 1990 s, which
is performed universally for the gramineae in particular.
At present, micro-dissection has been applied succe-
ssfully to isolate the egg cell of barley (Holm et al.,
1994), wheat (Holm et al., 1994; Kovacs et al., 1995),
maize (Kovacs et al., 1995), Rice (Zhao et al., 2000).
Zhao et al (2000) further attempted to compare the
effects of the ovule from the vertical profile, cross-
profile extrusion and separation of rice on egg and
found cross-sectional ovule very easy to maintain egg
cell in situ, to identify and to operate.
It is generally believed that dissection enzyme should
be used more in the plants (such as tobacco) with thin
beads heart, and easily separated ovules, while Micro-
dissection applies to plants (such as corn) with bulky
ovules and small numbers of ovules. The two methods
can complement each other, for example, in the sepa-
ration of rice and corn, the ovule of the short-term
digestion of the egg will help improve the efficiency
of separation, but if the enzyme is not thoroughly
cleaned or the over-all follow-up on the homozygous
which would be a negative impact on culture.
3 Egg, zygote and sperm in vitro culture,
sperm cells and egg cell fusion
The embryo sac cells which have been in vitro culture
include egg cell ,help cells, central cell, zygote, and
artificial insemination central cells. The most eye-
catching ones are egg cell and zygote in vitro culture.
Kranz et al (1995) first reported that the egg cell of
corn starts splitting and developing small cell mass in
high concentration of 2,4-D, but it can not further
grow. Subsequently, Zhao et al (2000) inducted the
first split in the cultivation of egg cells. Zygote can be
divided into homozygous and artificial zygote. Homo-
zygous zygote was easier to get than artificial zygote,
so homozygous zygote has been separated in many
species for studies. Homozygous of the four natural
crops, barley (Holm et al., 1994), wheat (Holm et al.,
1994; Kumlehn et al., 1998; Pónya et al., 1999), maize
(Leduc et al., 1996) and Rice (Han et al., 1998; Zhang
et al., 1999) in vitro culture have been able to
regenerate plants. Zhang et al (1999) found the homo-
zygous development of rice is not in accordance with
normal embryonic development mode in the cultivation
of rice’s homozygous, but generated plants after by
the bipolar embryoid or the formation of adventitious
root. Zhao et al (2002) used embryonic cell lines of
rice as suspension cell husbandries, obtained the
desired result in inducing the growth of zygote by
micro-feeding room culture, the rice zygote successfully
splits into multi-cell mass. Artificial zygote is induced
in vitro by a combination of methods. The methods by
which the egg and sperm cells were induced to form
artificial zygote are as following: electric fusion,
calcium-induced fusion, and PEG-induced fusion.
Zygote obtained from corn after sperm-egg fusion,
formed the plant in vitro culture through electric
fusion (Kranz et al., 1991). Faure et al (1994) put the
egg and sperm cells of the corn into 5 mmol/Lcacl 2,
and found that the sperm cells and the egg cell fused
in a very short time. By increasing the concentration
to 0.05 mol/L cacl2, the frequency of corn sperm-egg
fusion had also increased, but the zygote formed
multi-cell mass and no plant was obtained after the
fusion. Kovacs et al (1995) found slightly inequal split
in the cultivation artificial homozygous of wheat.
Such successful studies in vitro culture will form a
quality test system for genetic transformation and
improvement of crops.
4 Molecular biology research
The use of in vitro fertilization system in the process
of gene expression is a new field. The two major
technological bases of study have matured, they are,
access to low-volume manual egg cells and zygote,
and the method of building a micro-cell cDNA library.
RT_PCR application technology, 128 egg cells by the
separation of corn and 104 artificial zygote in vitro
fertilization after 18 hours of construction of cDNA
library, and a number of specific expression in the egg
or the fertilization induced expression gene were
isolated (Leduc et al., 1996). Gou et al (2001) first
built a CDNA library of rice sperm cells, and isolated
the expressional gene from the sperm cells. Bai et al
(2002) first isolated the full-length gene RSG6f from