Bottle gourd

1. BOTTLE GOURD
Submitted to
Dr. MAMTA PATHAK
Submitted by
MONISHA T

3. INTRODUCTION
 Bottle gourd: Lagenaria siceraria
 Diploid, 2n=2x=22 (x=5, n=5+5+1)
 Eulogized as ‘White flower gourd’
 An important multi-purpose cucurbit crop grown for its leaf, fruit, seed and also for
the hard shell which is ideal for making containers for decoration and other household
uses.
 Widely cultivated and used for human consumption in Sub-Saharan Africa (SSA)
providing vital human nutrition and serving as food security crop.
 Wide genetic variation among bottle gourd genetic resources in Africa for diverse
qualitative and quantitative attributes for effective variety design, product
development, and marketing.
 However, the crop is under- researched and -utilized, and improved varieties are yet
to be developed in the next generation of cultivars and commercialized in the region.

4. OBJECTIVES
 To provide progress on bottle gourd genetic improvement and genetic
analysis targeting agronomic and horticultural attributes, nutritional
composition, biotic and abiotic stress tolerance to guide current and future
cultivar development, germplasm access, and conservation in SSA and
globally.

5. PROGRESS IN BREEDING
Fruit morphotypes of bottle gourd accessions
cultivated in South Africa:
(A) BG-67, verrucose fruit texture and club-shaped
fruit
(B) BG-78, light-green fruit color and long neck
(C) BG-70, white-green fruit color
(D) BG-79, cultivated for its edible fruit,
(E) BG-100, commercially grown,
(F) BG-80, light-green fruit color, small-neck, and
corrugated skin texture,
(G) BG-27, dark and light green fruit color and
slightly curved fruit neck.
The accessions were originally collected from
various farmers’ fields, cross-checked and
compared with Lagenaria specimens maintained by
the Agricultural Research Council—Herbarium Unit
(South Africa). All collections were confirmed to be
bottle gourd.

6.  Genetic variability exists in bottle gourd genetic
resources for fruit horticultural traits useful for
strategic breeding and cultivar development.
 The crop show variation for fruit shape, size,
length, color, and texture.
 To develop “new” genotypes of bottle gourd,
crosses can be made between cultivars
expressing the traits of interest (e.g., neck
shape, neck length, fruit shape, and color).
 Selection of progenies showing the trait of
interest in the F2 generation to develop
desirable individuals.
1) FRUIT QUALITATIVE AND AGRONOMIC TRAITS
 Agronomic traits including flowering time, plant
height, no. of main and lateral branches per
plant, fruit weight, and no. of fruit per plant
show high levels of variation (Mashilo et al.)
 Eg: For direct improvement of fruit yield,
selection for high number of female flowers is
desirable due to high correlations between
these two traits.
 Hybrid varieties of bottle gourd may play a vital
role in satisfying the interest of producers and
consumers. The identification and utilization of
the most heterotic crosses is important for
hybrid breeding.

7.  The leaves are rich source of minerals and the fruit is vital source of amino
acids and the seed contains crude protein of 35%, crude lipid of 39%.
 However, the nutritional value of bottle gourd may be affected by the
presence of unfavorable anti-nutrients such as tannic acid, oxalates and
CUCURBITACIN.
 As bottle gourd fruits, seeds and leaves are rich in cucurbitacin E and I,
Cucurbitacin content varying from 2 to 12.5 mg/kg is reportedly toxic to
humans although they have various health benefits.
 Hence there is a need for a strategic breeding approach to develop
varieties with desirable cucurbitacin contents to reduce phyto-toxicity and
to enhance the use of bottle gourd fruit for medicinal purposes.
2) NUTRIENTS AND ANTI-NUTRIENTS

8.  Bottle gourd is moderately resistant to a number of viral and fungal caused
diseases such as Papaya ringspot, Fusarium wilt, Powdery mildew, Cercospora
leaf spot and Tobacco mosaic virus, cucumber mosaic virus (CMV), watermelon
mosaic virus 2 (WMV-2), and yellow mosaic virus (ZYMV).
 One of the four commercial rootstocks widely used in watermelon production
to improve resistance to soil-borne pathogens, improve fruit yield and quality
of grafted watermelon.
 Varieties such as Pusa Naveen, Pusa Santushti, Pusa Samridhi, and Pusa
Sandesh reportedly had high level of resistance to cercospora leaf spot.
 Hybrids developed in India display high levels of resistance against Fusarium
wilt (Dhillon et al. 2016).
 From these reports, there is higher possibility of developing highly resistant
gourd rootstocks/hybrids for watermelon production.
3) BIOTIC STRESS TOLERANCE

9.  Exhibits some level of drought tolerance and high salinity tolerance
compared to other cucurbits.
 Understanding of the physiological basis of drought tolerance in bottle
gourd can aid effective screening and identification of novel genetic
resources for breeding.
 Secondary metabolites such as cucurbitacins are produced in response to
drought and heat stress and may also be important indicators of stress
tolerance.
 The authors suggested that the accumulation of cucurbitacins may be a
potential physiological marker for identification and selection of bottle
gourd genotypes for drought tolerance breeding.
 Therefore, understanding the role of cucurbitacins for abiotic stress
tolerance may aid as a novel selection/breeding criterion for abiotic stress
tolerance breeding in bottle gourd or related cucurbit crops.
4) ABIOTIC STRESS TOLERANCE

10.  Wild species are a useful genetic resource for introgressing useful genes for biotic and
abiotic stress tolerance. They are rich in phytochemical compounds especially cucurbitacins
for pharmaceutical applications and can be used in Interspecific hybridization to transfer the
economically important traits to cultivated bottle gourd.
 Marker-assisted breeding is a complementary tool to phenotyping allowing accelerated
cultivar development and deployment.
 Molecular markers such as RAPD, SSR and SNP markers have been used to assess genetic
variability in bottle gourd and identified genetically distant genotypes for cultivar
development.
 Among these, SSR markers show high degree of polymorphism and are highly discriminative
of bottle gourd germplasm (Xu et al. 2011).
 To date, there is little information regarding genome size and chromosomal variation in
cultivated and wild species and dearth of knowledge about QTLs controlling qualitative and
quantitative traits, biotic and abiotic stress resistance in bottle gourd.
5) GENETICS AND GENOMICS

11.  Genetic engineering: Technique of inserting new genetic information into existing cells in
order to improve the expression of traits of interest.
 Agrobacterium tumefaciens-mediated transformation provided opportunities for
incorporating useful traits in bottle gourd.
 Incorporating CBF3/DREB1A genes (cold tolerance) and AVP1 genes (drought tolerance)
resulted in bottle gourd genotypes with improved abiotic stress tolerance and agronomic
performance.
 Genome editing: Potential to develop agronomically and nutritionally superior genotypes
with biotic and abiotic stress tolerance.
 CRISPR/Cas9 is the widely used genome editing platform allowing for substantial
improvement of economic traits.
 Genetic modification is an effective approach for creating genetic variation for desired traits
for bottle gourd improvement, however this approach has not been widely adopted.
6) GENETIC ENGINEERING AND GENOME EDITING

13.  Drought is one of the leading constraints affecting global crop production and
productivity.
 Under drought stress, key physiological responses including synthesis and accumulation
of compatible solutes which are referred to as osmoprotectants or osmolytes
responsible in lowering of the cell water potential and enhancing water extraction
capacity in water-limited environments (Ramanjulu and Sudhakar, 2000).
 Blum and Sullivan (1986) reported that plants constantly experiencing drought stress
may possess or develop some unique physiological drought adaptation mechanisms.
 Cucurbitacins are generally toxic to many organisms and therefore, their natural role in
plants is probably to act as a defense mechanism against pathogens and pests and they
set to accumulate during stresses.
 However, the level and role of cucurbitacins content in plants to drought stress are not
well-documented. Knowledge on their role and importance in drought adaptation may
contribute to the development of reliable selection criteria for drought tolerance
breeding.
INTRODUCTION

14. OBJECTIVES
 To determine the relationship between accumulation of cucurbitacins with
leaf gas exchange and chlorophyll fluorescence parameters in bottle gourd
under drought stress condition in order to establish the relationship
between cucurbitacins accumulation and drought tolerance.

15.  Plant materials: 12 landraces namely: BG-27, BG-31, BG-48, BG52, BG-58, BG-67, BG-70, BG-
78, BG-79, BG-80, BG-81 commonly grown under dryland conditions and a standard check
landrace “GC” grown with economically important traits.
 Experimental design and crop establishment:
i. Controlled pot experiments under glasshouse conditions.
ii. 12 × 2 factorial experiment laid under a completely randomized design with 3
replications. The 12 levels denominated bottle gourd landraces, while the 2 levels
represented watering regimes (drought-stressed [DS] and non-stressed [NS]
conditions).
iii. Plants under drought stressed condition were irrigated until the formation of six fully
expanded leaves and thereafter irrigation was withheld for 10 days before sampling.
iv. Plants in the non-stressed condition were watered daily to maintain soil moisture
content at approximately 40% (field capacity).
MATERIALS AND METHODS

16. PARAMETERS TAKEN
 Soil moisture content
 Gas exchange and chlorophyll fluorescence parameters
i. Stomatal conductance (gs), Net CO2 assimilation rate (A),
Transpiration rate (T), Intercellular CO2 concentration (Ci) and the
ratio of intercellular and atmospheric CO2 (Ci/Ca) concentrations.
ii. Maximum quantum efficiency of photosystem II photochemistry,
Effective quantum efficiency of photosystem II photochemistry
(ФPSII), Photochemical quenching (qP), Non-photochemical
quenching (qN), Electron transport rate (ETR).
 Determination and quantification of cucurbitacins E and I

17. HYPOTHETICAL MODEL

18. FINDINGS
 Cucurbitacin E and I were detected under NS and DS conditions in some of the
tested bottle gourd landraces.
 Significant and positive correlations were observed between cucurbitacin I
content with ETR/A.
 The current study identified drought tolerant bottle gourd landraces namely:
BG-48, BG-58, BG-70, BG-78 and BG-79 based of high values for gs, T, A and qN
under DS condition.
 These selections may be useful for drought tolerance breeding in bottle gourd
or related cucurbits.
 There is a need for further investigation whether cucurbitacin I accumulation
can be involved in the regulation of the physiological processes evaluated in
the present study.

20.  Heat stress negatively affects physiological processes, reproduction, and adaptation in crop
plants, which are caused by global climate change.
 Heat tolerance is a complex trait controlled by quantitative trait loci (QTL), which makes it
difficult to introgress multiple favorable alleles into recipient susceptible varieties.
 One method to mitigate abiotic and biotic stresses in vegetable production is grafting. For
example, bottle gourd has been used as the rootstock for watermelon to reduce heat stress
and improve performance of plant growth.
 By understanding the genetics of heat tolerance in bottle gourd and identification of DNA
markers may facilitate development of novel bottle gourd rootstocks for heat adaptation of
scion through marker-assisted selection.
 In this study, QTL-seq is used to identify major loci regulating heat tolerance in bottle gourd
based on REC.
 Putative candidate genes controlling heat tolerance and SNPs markers were identified using
the available genomic sequence for bottle gourd.
INTRODUCTION

21. P1 × P2
F1
F2
Phenotyping done in 3 experiments of F2 individuals including P1, P2 and 10 F1 plants
At 3 leaf stage, heat stress applied by moving the seedlings into 40°C temperature and 30000 lux for 6h
Relative Electrical Conductivity (REC) and Leaf Relative Injury (LRI) was measured
before and after heat treatment and ANOVA is determined
Selfing
P1= Heat tolerant L1 (P17)
P2= Heat sensitive L2 (P23)
MATERIALS AND METHODS
Crossing

22. 1) Genomic DNA isolation
Young leaf tissues of selected F2 individuals along with both the parents were isolated using CTAB
(Cetyltrimethylammonium bromide) method. Selected individuals were bulked to develop
contrasting heat tolerant and sensitive pools.
2) Preparation of Contrasting bulks (Based on phenotyping: Low and High LRI values)
Nine Heat tolerant individuals (T pool)
Nine Heat sensitive individuals (S pool)
3) Quality and quantity assessment of isolated DNA
Estimated by agarose gel electrophoresis and UV-Spectrophotometer
4) The high-quality clean reads from L1, L6, T-pool and S-pool were aligned and SNPs were detected
from the valid BAM file using the GATK “UnifiedGenotyper”.
5) To identify candidate regions for heat tolerance QTLs, the Δ(SNP-index) for all the SNP positions
was obtained by subtracting the SNP-index of T-pool from the S-pool. The statistical confidence
intervals of Δ (SNP-index) were plotted.

24. RESULTS AND DISCUSSION
 REC and LRI can be used as an indicator of heat tolerance, which exhibited recessive
inheritance.
 Seven heat-tolerant quantitative trait loci (qHT1.1, qHT2.1, qHT2.2, qHT5.1, qHT6.1,
qHT7.1, and qHT8.1) were identified with qHT2.1 being a promising major-effect
QTL.
 3 non-synonymous SNPs were identified which are potentially associated with heat
tolerance. These SNPs were located in the genes that may play roles in pollen
sterility, intracellular transport, and signal recognition.
 Association of the three SNPs with heat tolerance was verified in segregating F2
populations, which could be candidate markers for marker assisted selection for
heat tolerance in bottle gourd.
 These results revealed the novel region of 11.03 − 19.25 Mb on Chr 2 harboring
qHT2.1 that may provide the basis for further exploration and fine mapping of novel
genes associated with heat tolerance in bottle gourd.

25. THANK YOU