FIELD OF THE INVENTION
This invention relates to a process for the production of somatic
seedlings. This invention further relates to a process for the
production of somatic seedlings of sandalwood plants by using
plant cell for tissue culture technology.
BACKGROUND OF THE INVENTION
It is known in the art to produce somatic seedlings in culture
using mainly three different processes. In one process, pro-
embryogenic callus mass is placed on solid medium containing
hormones (H1) for somatic embryo induction. After about 4-6 weeks
somatic embryos are produced asynchronously with embryos at
different stages of development/differentiation. The mature
embryos are then transferred to a different medium having other
hormones (H2) in different culture vessel for germination,
thereby raising somatic seedlings.
The drawbacks of this process are that a majority of somatic
embryos (>90%) are asynchronous and abnormal; often joined with
one another and very low number of normal somatic embryos are
obtained per gram of pro-embryogenic callus.
A second method known in the art for somatic seedling production
is direct somatic embryo production from any plant part, on solid
medium in presence of one hormone (H1). These embryos are then
germinated in a different medium in presence of another hormone
(H2) to produce somatic seedlings. This method suffers from the
drawbacks of poor response of plants to embryogenesis, ie. very
few plants respond to direct embryogenesis, and that a number of
embryos produced is very low whereas time taken is very long.
Yet another process known in the art is that pro-embryogenic
callus mass is cultivated in liquid medium in bioreactor in
presence of hormone (H1) to produce somatic embryos. The produced
embryos" are then germinated in solid medium in presence of
another hormone (H2). Even through the total number of embryos
produced by this process are higher than those produced by the
first two processes greater than 30% are abnormal.
In all of the above three processes, the total time taken from
embryogenic callus mass to somatic seedling production is greater
than six weeks. The residual effect of hormones H1 and H2 on the
subsequent stages are the main reasons for major developmental
abnormalities. The somatic seedlings which are apparently normal
do not survive in the field due to above mentioned abnormalities.
No process has so far been described to take care of the precise
exposure time of H1 and H2 and to prevent carry forward effect of
H1 and H2 to the subsequent stages.
OBJECTS OF THE INVENTION
It is therefore an object of the present invention to propose a
novel process for the production of somatic seedlings rapidly by
proper programming of incubation time of the embryos.
It is a further object of this invention to propose a novel
process for the production of somatic seedlings which gives
greater yield of the seedings from pro-embryogenic cell and is
therefore cost-effective.
Another object of this invention is to propose a novel process
for the production of somatic seedlings which is completed
aseptically in one bioreactor without any sub-culture.
DESCRIPTION OF THE INVENTION
Thus according to this invention is provided a process for the
production of somatic seedlings of plants comprising innoculating
a plant pro-embryogenic cell/callus mass in a nutrient medium
containing H1 hormones in a bioreactor,
draining out the spent medium,
adding fresh nutrient medium into the bioreactor followed by
cultivation of the embryos therein,
adding H2 hormones into the medium and allowing the somatic
embryos to germinate to somatic seedlings,
draining out the H2 hormone containing medium and adding fresh
nutrient medium containing an inhibitor into the bioreactor,
allowing the seedlings to develop in the medium to obtain the
somatic seedlings.
In accordance with this invention, an agitated bioreactor is
innoculated using plant pro-embryogenic cell/callus mass in a
first liquid medium containing H1 hormone(s) favouring somatic
embryo induction and maturation upto cotyledonary stage. The
agitation in bioreactor is provided in such a manner so that the
developing embryos move characteristically with their shoot and
root apices oriented acropetally and basipetally respectively,
for most of the time and thereby favour normalcy in development.
The nutrient medium is charged by aseptically pumping out the
spent medium and pouring in fresh nutrient medium without any
hormone. The somatic embryos are cultivated in this hormone free
(H zero) condition for 2-5 days depending on the plant species.
This step prevent residual effect of H1, which interfere with
next step hormone for germination. The yield of normal embryos
increase significantly because of this step.
The hormone(s) required for germination (H2) of somatic embryos
are added to the bioreactor, after filter sterilization. The
somatic embryos germinate to somatic seedlings. Thereafter, the
H2 containing medium is pumped out from bioreactor and fresh
medium with an inhibitor (I) for H2 is added to the bioreactor.
The germinated somatic seedlings are kept in this inhibitor
containing medium which eliminate the residual effect of H2 and
favour proper development of somatic seedlings. Such seedlings
exhibit much higher survival during green house hardening.
The nutrient medium is a liquid medium used for embryogenesis
such as the MS plant tissue culture medium proposed by Murashige
and Skoog (1962), according to Gamborg et al (1968), McCown plant
tissue culture medium proposed by Lloyd & McCown (1981) or CCC,
ie. chlorocholine chloride.
The hormones used for the culture are H1 and H2 where H1 is used
for somatic embryo induction and maturation and is selected from
auxius cytokenins etc.. The hormone H1 is added to the medium in
a proportion of (3.2-2.5 ppm.
The hormone H2 induces germination of the somatic embryos and is
selected from gibberellins or gibberellin - cytokinin mixture. H2
is used in a proportion of 0.2 to 1.5 ppm. The The inhibitor is used
to minimise the residual effect of H2 and is selected from
abscisic acid, ancymidol or chlorocholine chloride singly or in
combination. The inhibitor is used in a proportion of 0.2 to 1.0
ppm or 1.0 mg per litre of medium.
DESCRIPTION OF THE ACCOMPANYING DRAWIN6S
The invention will be apparent from the description with read in
conjunction with the accompanying drawing where Fig.l depicts a
schematic flow diagram of the process for somatic seedling
production according to the invention. Step 1 shows innoculation
of the bioreactor with the pro-embryogenic friable cell/callus
mass as innoculum.
In step 2, the first medium is replaced with a second medium
without any hormone, followed by cultivation in the hormone free