Introduction: For the past four decades of the 20th century, agriculture has benefited from the use of semidwarf phenotypes in many different crops. The use of these genes was one of the main driven forces of the so called ‘Green Revolution’ that resulted from the adoption of new semidwarf varieties and cultivation methods. The major impact was in cereal crops, first in wheat and then in rice. The semidwarf wheat varieties had improved harvest index from the increasing of grain yield at the expense of straw biomass and were more lodging resistant, thus supporting higher fertilizing rates. Besides that, because of the involvement of the dwarfing genes with the gibberellin biosynthesis, fundamental research has been conducted with dwarf mutants involved in the GA biosynthetic pathway. Dwarfing genes have been instrumental in dissecting the gibberellins (GAs) biochemical pathway in some species and in elucidating the plant elongation process. CONCLUSION: • The dwarfing genes are considered to be a main driving force of the green revolution, which resulted from the adoption of semi-dwarf cultivars in the past. There is a potentiality still lies in other agricultural important crops with respect to exploration of dwarfing genes. • Beneficial effect of mutation in HvGA20ox2 gene for sdw1.d allele associated in barley with lodging resistance leads to reduction in flowering time but maintained yield, without affecting grain quality traits and thousand grain weight • Accidental discovery of new allele of Bna.IAA.C05 responsible for the dwarf mutant generated from tissue culture may provide valuable genetic resource for B.napus. The mutation was associated with downregulation of auxin related genes including small SAUR genes in dwarf mutant • Crispr/cas-9 system could be successfully employed for development of semi-dwarf-1 (sd-1) mutant in rice landraces. The breeding in combination with genome editing techniques is an effective way to improve agronomical characteristics and to harness the genetic potential of landraces. • Quadruple mutant of GmLHY gene developed through genome editing reduces plant height, shorten internodes. The significance downregulation in GA metabolic pathway genes suggested that GmLHY encodes MYB-TF regulates plant height through GA mediated pathway. • Dwarfing genes in combination with elite background could be a strategy for the next-generation plant breeding to break the yield plateau of agriculturally important crop varieties.

1. NAVSARI AGRICULTURAL UNIVERSITY
TOPIC: Dwarfing Genes in Crop Improvement
Subject: MBB 591
Master’s Seminar
Presenter
SOJITRA AJAY MANSUKHBHAI
2010120104
Department of Plant Molecular Biology and Biotechnology
ADVISOR:
DR. VIPULKUMAR B. PAREKH
Assistant Professor(Biotechnology)
Department of Basic science and Humanities,
College of Forestry
ACHF, N.A.U., Navsari.-396 450.
CO-ADVISOR:
DR. K.G. MODHA
Associate Professor (GPB)
Department Of Genetics And Plant Breeding,
N.M. College of Agriculture
N.A.U., Navsari.-396 450.
WELCOME TO SEMINAR SERIES 2021-22

2. Content will be discuss….
Dwarfing gene in plants : Introduction
Throwback :Dwarfism played vital role in Green Revolution
Gibberellin:The reason behind the plant height
Gibberellin biosynthesis pathway
Types of dwarf-Mutant
New Plant BreedingTechnologies
Case -studies
Conclusion
2

3. 3
Dwarfing: “It is a process in which, breed cultivar of plant become significantly smaller than
the standard member of their species.”
Role of dwarfing genotype: Within important crops, such as wheat and rice,
The Green revolution brought about significant increases in yield by combining the breeding of high yielding dwarf
varieties with agricultural mechanization and fertilizer application.
Extreme dwarf varieties are usually correlated with poor agronomic performance and traits such as smaller grains,
excessive tillering, or narrow leaves therefore semi-dwarf varieties are used to enhance yield and lodging resistance.
In rice, the semi-dwarf gene sd1, which regulates a key step in gibberellic acid (GA) biosynthesis, has been used
worldwide in rice production.
The use of Rht (Reduced height) genes, involved in GA signaling transduction, was instrumental in bringing about
the “Green revolution” in wheat as well as other crops.
Dwarfing gene: Foundation of ‘Green revolution’

4. Epoch of the dwarfism or dwarfing gene:
“Dominance of tall allele over dwarf ” – Mendel, 1866 'Versuche über Pflanzen-Hybriden’
“ Le - Stem internodes” – White,1917 ‘Studies of Inheritance in Pisum.’
“Characters of tallness and dwarfness is depends on the endogenous Gibberellic acid mechanism in plants.”– Brian and
Hemming,1958 ‘Effect of Gibberellic Acid on Rate of Extension and Maturation of Pea Internodes’
4

5. DARUMA FULTZ
DARUMA /
FULTZ
US TO JAPAN
DARUMA / FULTZ TURKEY NORIN-10
History: Green revolution in wheat
In 1917, wheat genotypes
imported US to Japan
In 1925, Japanese breeder crossed with Russian
turkey variety
In 1935, this combination
released as Norin-10 5

6. 6
Story behind the Green Revolution
S.C. Salmon at Japan Japan to US
Vogel Norin-10 × AmericanVariety Gaines
Intense selection of progeny
Norin-10 × Brevor 14
Characteristics are as follows:
• Higher number of tillers,
• Higher lodging and shattering
resistance
• Medium spikes,
• Medium plant height.
Lines sent to CIMMYT,
Mexico.
Lerma-rojo and Sonara-64

7. Taiwan breeding program
TN-1
Philippines breeding program
MIRACLE RICE
World-wide multiple crosses with local
varieties- indica, japonica
7
Chow-wu-gen ×Tsai-Yuan-Chunj Dee-gee-woo-gen × Peta
Discovery of rice semi-dwarf : Green revolution
IR-8

8. Gibberellic acid: Hormone of the stem length
Extracted GA from Plant
tissues
Gibberellic acid (also called gibberellin A3, GA, and GA3) is
a hormone found in plants and fungi.
Its chemical formula- C19H22O6.
Purified form- White to pale-yellow solid.
Gibberellic acid pathways play vital role at 4 stages of plant
development
1. GA signaling in Dormancy and seed germination.
2. GA biosynthesis and signaling in the apical meristem
3. GA in the Flowering and sex expression
4. GA in the embryo development
8

9. 9
YABUTA in 1935 Isolation of gibberellin from the Gibberella fujikuroi
Takahashi in 1955 Isolation of its methyl ester named as Gibberellin A1 ,A2 and A3.
Brian and Hemming in 1958 Identified that dwarf pea plant does not contain GA3
Yamaguchi in 2003
Bioactive GA1 is found in most higher plants species;
GA4 major bioactive GA in Arabidopsis thaliana
History: Gibberellin research

10. Conversion of ent-kaurene to GA12
via microsomal cytochrome P450
monooxygenases
Production of ent-kaurene
in proplastids
Formation of C20-and C19−GAs in
the cytoplasm
Figure:1 Gibberellin Biosynthetic path 10

11. 11
Types of Mutants
Note: In hexaploidy wheat, dwarfing has been achieved mainly through the introduction of the mutant
Reduced height-1 (Rht-1) genes that encode with DELLA proteins, which act to repress GA-responsive
growth.

12. Biotechnological approaches to manipulate plant’s height
Molecular markers
Gene silencing
Genome editing techniques
QTL mapping
Marker assisted selection (MAS)
Transgenic approach
Biotechnological
approaches
12

13. CASE STUDY-1
Teplyakova et.al 2017 Almaty,Kazakhstan
13

14. Background and Purpose of this study
Morex (Tall) Sdw1 cv. Berke (Semi-dwarf)
Sdw1/denso allele
(DHL- double haploid lines)
(Morex/berke)
-----------7BP deletion-----------
Aim:
• Estimation of the pleiotropic effect of sdw1/denso allele in two different
environment by applying QTL approach.
• Evaluation of plant’s height, heading date, thousand grain weight.
14

15. Modus operandi
Seeds of double haploid lines (DHLs) derived from the cross between Morex and Barke.
Field trials of the Morex/Barke DHLs population were conducted in two experimental stations of
Vir: in Pushkin ( North of Russia) and Krasnodar(South of Russia).
DHLs were evaluated :Heading date (HD)
Plant height (PH)
Thousand grain weight (TGW)
15
QTL MAPPING
CAPS assay to reveal 7 bp deletion in exon 1 of HvGA20ox2 gene

16. Fig.1- Re-sequencing and mapping of HvGA20ox2 gene in
Morex/Barke DHLs population
Re-sequencing of exon-1 of
HvGa20ox2 gene for Berke,
Triumph, Franklin
Primer was designed based on
published contig of Morex for
HvGa20ox2
Sequence found as 488 bp for
all varieties and 100% derived
from the sdw1/d allele
7-bp deletion leading to shift of
reading frame in Berke,
Triumph, Franklin varieties
The intact and altered proteins
have just 34 amino acids are
common, followed by complete
different polypeptides
16

17. Fig.-2 Mapping the HvGA20ox2 gene by CAPS marker.
Table 1: Mapping of the HvGA20ox2 gene in the Morex/Berke DHLs
17

18. 7-bp deletion in exon 1 of hvGA20ox2 associates with plant height segregation in Morex/Borex
DHLs population
Fig.3 : Mapping of plant height in Morex/Barke DHLs population on QTL
18

19. • The remarkable difference in flowering time is non-significant, Morex/Barke a recessive allele of Ppd-H1
gene was reported, suggesting reduced response to the long day.
• QTL varied from 28% to 41% in two geographical locations.
• DHLs, inheriting sdw1/denso allele from Barke, initiated flowering 3-5 days later than DHLs carrying the
HvGa20OX2 allele from Morex.
Location -Pushkin
Location -Krasnodar
Fig. 4 QTL mapping for Ppd-H1 gene at two different location
19
7-bp deletion in exon 1 of hvGA20ox2 associates with Heading date segregation in Morex/Borex
DHLs population

20. Fig-5: Variation of thousand grain weight (TGW) and grain quality traits in Morex/Barke DHLs
population depending on 7-bp deletion in exon 1 of HvGA20ox2 gene
• QTL peak for the trait was consistently mapped on 2H
chromosome (77.6 cM)
• Co-segregating with SNP 3_0896,
• POPA SNP marker of the VRS1 locus controlling the
inflorescence type.
Location -Pushkin
Location -Krasnodar
20
In the Morex/Barke cross
• Two-rowed parent Barke carries the dominant
Vrs1.b3 allele,
• Six-rowed parent Morex has the recessive vrs1.a1
allele.
• The two-rowed DHL offspring showed higher
TGW values compared to six-rowed DHLs.
(77.6 cM)

21. Conclusion – Case study 1
• In this study the 7-bp deletion in HvGA20ox gene, which was recently proposed as the functional polymorphism
of sdw/denso locus in barley, was significantly associated with reduced plant height and delayed flowering
time in the segregating population of DHLs derived from cross of medium tall barley Morex and semi-dwarf
sdw1.d (sdw1/denso) variety Barke, independently on environment cue.
• Study of grain yield and grain quality there is none the another effect was found in the test after that based on the
result there is no pleotropic effect on the seed.
• On other hand, the sdw1.d allele in barley seems does not relate directly to the grain yield potential, but is
associated with lodging resistance.
21

22. CASE STUDY-2
Cheng et.al 2019 Gansu, China.
22

23. one dominant dwarf mutant was identified during the genetic transformation in B. napus.
Background and Purpose of this study
How did that mutation generate in oilseed rape?
Evaluation phenotypic characteristics of new generated dwarf.
23
Mark the responsible gene on the chromosome
Brassica napus
A. thaliana mir169d
100 sample in
tissue-culture
One dominant mutant found

24. Modus operandi
• Plant materials and phenotyping for plant height
1. The B. napus dwarf mutant was originally isolated from tissue culture.
2. A cross was made between 48557(wild type, female parent) and the G7 dwarf (pollen donor) to create
F1 plant
3. Which were subsequently self to create a segregating F2 population.
4. Plant height was measured when plants attained maximum height at the final flowering stage.
24
• Expression analysis of dwarfing candidate gene by semi-quantitative RT-PCR and RT-qPCR
• Microscopy analysis
• Mut-map analysis
• RNA-seq analysis of differential gene expression
• KEGGS analysis

25. CONSTRUCTEDVECTOR
Fig.-6: Validating T-DNA region in the accidently developed mutant- G7
25

26. Fig-7: Comparative analysis of characteristics of accidental identified
dwarf mutant:
A B
C
D E
26
G7(˜30 cm)
WT (˜150cm)

27. Inheritance of dwarf phenotype
WT
(48557)
G7
F1
F2
F2 population: 255
G7 plants :190
WT plant: 65
27
Fig-8 : Distribution of plant height in the F2
population derived from the cross of G7 and 48,557

28. 28
Genomic DNA sequenced: data generated as
follows:
G7: 510,946, 104 (0.51 GB)
48557: 523,601,254 (0.52 GB)
Dwarf-pool: 438,302,148 (0.43 GB)
Tall-pool : 411, 082,480 (0.41 GB)
unique mapped reads :
G7: 232,279,673
48557: 215,670,613
Number of SNP:
G7:53,888,50
48557: 53,709,65
Sub genome A and C
Fig-9: Identification of candidate genomic
region by MutMap

29. Fig-10: Candidate identified by MutMaP
BnaC05, SNP resulting in amino acid variation were
identified within 18 candidate genes.
Putative gene function prediction revealed the candidate
genes had been implicated in the control of plant architecture
BnaCo529300D, a homolog of AtIAA7 in Arabidopsis.
The SNP in Bna.IAA7.C05 within conserved domain II core
sequence change from GWPPV to EWPPV.
29

30. Fig- 11: Dwarf phenotype co-segregating with the dCAP marker for SNP variation in
Bna.IAA7.C05
30

31. 31
9516 gene
GO analysis
cellular metabolism genetic environment organismal
KEGGS analysis
Fig-12: RNA-sequence analysis in whole genome; DEG analysis; KEGGS analysis

32. 32
Fig-13: Heatmap analysis for IAA gene expression; SAUR(small auxin up-regulation)
responsive gene analysis

33. Conclusion – Case study 2
• Mutant with dwarfism and down-curved leaf was isolated from tissue culture processes. The candidate region for
dwarfism was mapped to a 0.6Mb region of B. napus chromosome C05 through the MutMap method.
• Further candidate genes analysis revealed that one amino acid substitution from G to E in the conserved motif
GWPPV of Bna.IAA7.C05 lead to dwarfism phenotype. This mutation of Bna.IAA07.Co5 resulted in decreased
expression of other IAA and auxin response genes expression.
• Findings identified one new allele of Bna.IAA7.C05 responsible for plant dwarf phenotype and provide insight for
understanding a dominant dwarfism mutant in B. napus
33

34. Case study -3
Xingming Hu et.al. Hangzhou, China
2019
34

35. Background and Purpose of this study
35
Aim : Relieving genetic erosion and improving the yield of modern rice varieties to satisfy food supply demand.
To show that using gene editing on available landraces can rapidly increase genetic diversity and produce
new varieties that satisfy current production requirements.
Crispr/cas-9 Genome editing sd1
Kasalath
Characteristics of Kasalath;
• Tolerance to low phosphorus
• Inbuilt insect-pest resistance
Develop new lines
Sd1-1kas,Sd1-2kas,Sd1-
3kas,Sd1-4kas,Sd1-5kas

36. Modus operandi
36
• Plasmid construction and Plant transformation
• Seed germination rate measurements
• GA- treatments
• N-fertilizer treatments

37. Construction of CRISPR/cas-9
37
Fig-14: Construction map of the Crispr/cas-9 along with gRNA and Promoter

38. Fig-15: Validation of Binary vector in the new generated Sd1 lines
38

39. Note: In the gRNA1 and gRNA2 targeting site in Kasalath the mutation was 44% and 68% respectively.
39
Fig: 15- Sequencing analysis for the new developed lines

40. 82.5 87.62 87.16 91.5
40
Kasalath Sd1-1kas Sd1-2kas Sd1-3kas Sd1-4kas Sd1-5kas
Fig:16: phenotypic comparison of the new sd-1 lines with their parental line

41. 41
Table-2: Agronomic Comparison of new Sd-1 lines with their Wild type

42. • HN: high nitrogen-20 kg N ha−1.
42
• N: No Nitrogen,
• LN: Low Nitrogen-8kg N ha−1,
• MN: moderate Nitrogen-14 kg N ha−1
Fig-17: Phenotypic comparison of new sd-1 line for NUE

43. 43
Table:2 comparison of agronomic characteristics between new Sd-1 lines for NUE
Pass

44. 44
Fig-18:Yield measurement comparison for NUE
Note: Plot size was 10m * 5m

45. 45
b
Fig-19: GA application comparison

46. Note:
A. Total root length and surface area of Kasalath, sd1-3kas, in low P(0.5 mg/L), Check(10 mg/L) and high P (25
mg/L) hydroponics solution of 20 days
B. The root architecture of Kasalath, sd1-3kas, and sd-1-5 kas plants under different concentration of P. 46
Fig-20: Inspection of Root with Phosphorus

47. • The Crispr/cas-9 editing technique can be use to more rapidly create the sd-1 mutation in desirable germplasm
• Creating additional sd1 alleles in other desirable landraces could help to improve genetic diversity in rice and
benefit rice production.
Conclusion – Case study 3
47

48. Cheng et.al. 2019 Guangzhou, China
48

49. Background and Purpose of this study
Aim:
• Generate the transgene soyabean plant with quadruple mutant: Gmlhy1a1b2a2b
• Aim to generate transgene-free plant by application of mendelian inheritance law
• Evaluation of the new developed transgene plant on the basis of phenotypic characteristics
49
CRISPR/Cas9 GmLHY T-2 generation Shorter then WT
Length Elongated
Hypocotyl

50. Modus operandi
• Plant material, growth condition
50
• Plasmid construction
• Stable soybean transformation
• Agrobacterium rhizogenes-mediated transformation of soyabean hairy roots
• Identification of indued mutation using PCR and sequencing analysis

51. Fig-21 Target site selection
51

52. Crispr
vector
Cas 9
Driven by the CaMV355
promoter
4 gRNAs
52
Fig-22: construction map of Cas-9 for GmLHY

53. Study the function of the four genes
Note:
4- target adopter were used including target 1 and 2 for,GmLHY2a and GmLHY2b and target 3 and 4 for GmLHY1b
GmLHY1a.
53
Fig-23: Identification of location of target mutant in gene sequence

54. Fig-25: Stable soybean transformation using Crispr/cas-9
54

55. 55
Fig-26: Developing of T1 generation from T0-7 lines

56. 56
Fig-27: examine T2 generation for phenotypic characters

57. 57
Fig: 28 Diurnal expression

58. 58
Fig-29: examine T2 generation for GA sensitivity
Uniconazole
Uniconazole

59. 59
Fig-30: Development of dCAPs marker for GmLHY1a1b2a2b

60. Conclusion – Case study 4
• The Crispr/cas-9 system can be used for multiplex gene editing to advance crop plant breeding, In the
present study, they used Crispr/cas-9 –based multiple genome editing to successfully obtain quadruple
mutant of GmLHY in soybean.
• Further, this results suggested that GmLHY directly or indirectly improves the expression level of GA
synthetic genes and GA response- related genes to regulate soybean plant height.
• This findings offer a good approach to use of the genome editing to generate non-transgenic soybean
genotypes and provide insight into the mechanism underlaying plant height regulatory network in crop plant
species.
60

61. Seminar conclusion
61
 Quadruple mutant of GmLHY gene developed through genome editing reduces plant height and shorten
internodes. The significance downregulation in GA metabolic pathway genes suggested that GmLHY encodes
MYB-TF regulates plant height through GA mediated pathway.
 The dwarfing genes considered to be a main driving force of the green revolution, which resulted from the
adoption of semi-dwarf cultivars in the past. There is a potentiality still lies in other agricultural important crops
with respect to exploration of dwarfing genes.
 Beneficial effect of mutation in HvGA20ox2 gene for sdw1.d allele associated in barley with lodging resistance
leads to reduction in flowering time but maintained yield, without affecting grain quality traits and thousand
grain weight
 Accidental discovery of new allele of Bna.IAA.C05 responsible for the dwarf mutant generated from tissue
culture may provide valuable genetic resource for B.napus. The mutation was associated with downregulation of
auxin related genes including small SAUR genes in dwarf mutant
 Crispr/cas-9 system could be successfully employed for development of semi-dwarf-1 (sd-1) mutant in rice
landraces. The breeding in combination with genome editing techniques is an effective way to improve
agronomical characteristics and to harness the genetic potential of landraces.
Dwarfing genes in combination with elite background could be a strategy for the next-generation plant breeding
to break the yield plateau of agriculturally important crop varieties.

62. 62
Future thrust
• Exploitation of dwarfing genes along with next generation molecular breeding techniques in
combination to elite background will provide opportunity to test ‘mix and match’ for
possibility of next-green revolution.
• Possibility of tailor made dwarfing gene/allele could be check through genome editing in GA-
pathway,TF-regulation, epigenetic regulation, etc. in crop plants.
• Regulation of degree of dwarfism with help of modern gene regulation techniques like,
synthetic biology, nano biotechnology, promoter engineering etc. may open-up the new
avenues for the customized crop improvement.
• Possibility of speed-breeding coupled with temporary dwarf plant statures may hasten-up the
breeding cycle as a strategy for next-gen plant breeding.

63. 63