PhD

Black pepper (Piper nigrum L.) universally honoured as “Black Gold” and
also known as “King of Spices” is one of the most important spices in the world.
It is a perennial climber belonging to the family Piperaceae and is valued
throughout the world for its spice value and medicinal properties. Black pepper is
grown in Southern India mainly in Kerala. Here productivity is declining due to
various reasons and continuous vegetative propagation leads to accumulation of
diseases and finally results in unproductive vines. Therefore, use of quality
planting material of improved varieties is necessitated for the enhancement of
productivity. Apomixis is a mode of asexual reproduction where the sexual organs
are utilized, but the seeds develop without fertilization. It combines the advantage
of seed propagation and vegetative propagation, and can be utilized for
developing disease free planting material without losing the clonal integrity. The
present study on “Validation of apomixis and transcriptome analysis for detection
of the genes related to apomixis in black pepper (Piper nigrum L.)” was
undertaken at the Centre for Plant Biotechnology and Molecular Biology, College
of Horticulture, Vellanikkara during the period 2011-2014 with the objective to
validate apomixis in black pepper varieties, Panniyur-1 and Panniyur-2 through
controlled pollination studies and to identify differentially expressed genes
associated with apomixis through transcriptome analysis.
Studies were conducted on bush pepper plants of the selected varieties
maintained in the green house. Floral biology of Panniyur-1 and Panniyur-2 were
studied. The female phase in Panniyur-1 started 8th day after spike initiation and
completed on 27th day. Stigma became receptive on 9th day and receptivity was
indicated by creamy white colour. Beginning of male phase indicated by anther
emergence on either side of the ovary occurred on 19th day of spike emergence
and active dehiscence of anthers occurred on 20th day. In Panniyur-2, beginning of
female phase started on 14th day after spike initiation and stigma became receptive
on 15th day. Beginning of male phase occurred on 22nd day of spike emergence
and active dehiscence of anthers occurred on 23rd day. So in Panniyur-1 and
Panniyur-2 the active female and male phase is separated by 10 and 8 days,
respectively.
Attempts were made to confirm apomixis in Panniyur-1 and Panniyur-2
varieties by allowing berry development in controlled condition by bagging of
spike initials. Berry development occurred under bagged condition. In this study,
upper six berries were considered as apomictic and lower six berries were
considered as pollinated. Efforts were made to regenerate apomictic and non-
apomictic progenies under in vitro and ex vitro. In vitro embryo culture resulted in
embryo germination and multiple shoot induction in SH medium with hormones.
But plants were lost due to microbial contamination and phenolic interference. So
ex vitro germinated seedlings were used for further studies.
Histological examination of pollinated and unpollinated ovaries was done
through microtomy. The results revealed that in case of pollinated berries, the
sexual fertilization occurs and embryo develops inside the embryo sac in the
micropylar end. Whereas, in apomictic embryo sac more than one aposporous
initial cells were observed which were arising from a somatic cell located in the
nucellus. So, it can be concluded that facultative apomixis exists in black pepper
varieties P1 and P2 and the embryo develops parthenogenetically.
The molecular characterization of apomictic and non-apomictic seedlings
was done through Inter Simple Sequence Repeats (ISSR) and Simple Sequence
Repeats (SSR) assay. Isolation of good quality genomic DNA from apomictic,
pollinated progenies and mother plant was carried out from the young leaves
using modified Rogers and Bendich (1994) as reported by Mogalayi (2011). In
ISSR and SSR assay certain primers showed polymorphism among mother plant,
apomictic and pollinated progenies. Similarity matrix was calculated and
corresponding dendrogram was also constructed for both markers using UPGMA
cluster analysis for P1 and P2 varieties. In ISSR assay the similarity coefficient
between P1 mother plant and apomictic progenies ranged from 69 to 89 per cent
while it was 64 to 66 per cent between mother plant and pollinated progenies. In
P2 variety similarity coefficient between mother plant and apomictic progenies
ranged from 76 to 90 per cent while it was 62 to 69 per cent between mother plant
and pollinated progenies. In SSR assay similarity coefficient between P1 mother
plant and apomictic progenies ranged from 50 to 100 per cent while it was 71 to
100 per cent between pollinated progenies and mother plant. Similar results were
obtained for P2 variety also. So apomictic progenies may differ from mother plant
in certain characters.
RNA mediated transcriptome analysis of the apomictic and pollinated
berries was done to detect differentially expressed genes. Good quality RNA was
isolated by modified LiCl precipitation method. Total RNA from the apomictic
and pollinated berries were taken for DDRT-PCR analysis. The first strand cDNA
was
synthesized
from
the
above
RNA
samples
using
HT11C
(AAGCTTTTTTTTTTTC). Each first strand cDNA was used for the second
strand amplification with 8 different arbitrary primers. The PCR product was
resolved in 6 per cent denaturing urea polyacrylamide gel and visualized after
silver staining. The differentially expressed cDNA fragments were retrieved from
the gel and reamplified and electrophoresed. The agarose gel electrophoresis
showed 7 transcript derived fragments (TDFs) ranging from 200-600 bp. TDFs
were cloned using pGEMT vector and were sequenced by outsourcing. The
sequence data were analysed by BLASTn and BLASTx. The sequences showed
homology to NADH dehydrogenase subunit J, acetyl-CoA-benzylalcohol
acetyltransferase and purine permease 4 but no significant sequence similarities
for apomictic genes deposited in NCBI database were found.