Download introduction

Biological Interventions for Enhancing Saffron Productivity in
Kashmir
F.A. Nehvi, S.A. Wani, S.A. Dar, M.I. Makhdoomi, B.A. Allie, Z.A. Mir
K. D. Research Station
Sher-e-Kashmir University of Agricultural Sciences & Technology
Kashmir
India
Keywords: Crocus sativus, in vitro micropropogation, mutation, variability
Abstract
The heterogeneity found in the natural saffron population for
morphological, developmental and yield component traits are primarily due to
the genetic and environmental factors. Development of high yielding genotypes
using the existing gene pool of saffron applying the biological tools shows
potential for improving the productivity of this crop. A survey undertaken to
study the extent of variation revealed wide spectrum of variability in saffron
flowers and corm samples collected from saffron growing areas of Kashmir, thus
implying a great scope for saffron improvement. Flowers completely devoid of
style & anthers and freaks with 4-5 stigmas where observed from the natural
populations. Collection and evaluation of saffron clones revealed possibility of
increasing yield per se and has lead to the identification of ten elite clones with
distinct yield superiority. Due to absence of sexuality mutation breeding
approach is another way of creating genetic variability in saffron. An attempt
was made to create new variants for economic characters through the induction
of mutation using Gamma irradiation. Existing saffron population was
irradiated with gamma rays from a co60 source at 0.25, 0.50, 0.75 and 1 kr doses.
Delayed sprouting was observed in higher dose (1 kr). Higher dose was also
associated with decreased corm yield and dry pistil weight/plant due to strong
reduction in percentage of flowering plants and number of flowers / plants.
Tissue culture studies have shown possibility to regenerate saffron plantlets and
in vitro corm development through somatic embryogenesis. Evaluation of in
vitro corms under field conditions revealed that corm development and survival
is directly proportional to initial corm weight. Survival percentage among invitro corms having average corm weight less than 1 g was only 26 %, whereas, it
was above 88 % in above 1 g corms. Weight gain after one year was 0.25 to 3.54
g.
INTRODUCTION
Saffron the legendary crop of J & K state has been under cultivation for so
many centuries. In spite of extremely high demand and value in the market for its
multiple uses in diverse fields as a dye, in perfumery, in food flavouring, limited
attention has been paid for its genetic improvement. Meiotic abnormalities result in
abnormal chromosome assortment and formation of an abnormal number of
genetically unbalanced spores which vary in shape and size leading to complete
sterility (Chichiricco, 1990). Therefore, utilization of heterogenity in the natural
population which is due to genetic and environmental factors offers a tremendous
scope for saffron improvement. Another approach for increasing variability for
selection through irradiation has not received much attention. The use of mutagenesis
could increase the natural variability for important characters such as high content of
active principles per stigma, variability in morphological and flower component traits
and precious emergence of flowers. Since the saffron reproduce only vegetative by
the corms any attempt to modernize saffron cultivation will therefore require efficient
mass production of pathogen free corms. Micro propagation of saffron has therefore
been advocated to be the best alternative for its propagation (Georage et al., 1992;
Ahuja et al., 1994). Present study was thus undertaken keeping in the view the basic
need of enhancing saffron productivity in Kashmir involving biological intervention.
MATERIALS AND METHODS
Present study was carried out at Saffron Research Sub-Station Dussu Konibal
Pampore a constituent Research Station of Sher-e-Kashmir University of Agricultural
Sciences & Technology of Kashmir located at 34.1oN latitude, 74.89o E longitude
with an altitude of 1650 m.a.m.s.l.
Extent of Variability and Identification of Elite Clones
438 flower samples each containing 100 randomly selected flowers were
collected from the saffron growing area of Jammu & Kashmir during the flowering
period (October-November). Parameters of variability were recorded for various floral
and quality traits viz., fresh flower wt (g), fresh Perianth wt (g), fresh anther wt (g),
fresh pistil wt (g), dry flower weight (g), dry perianth wt (g), dry anther wt (g), dry
pistil weight (g), stigma length (cm), style length (cm), pistil length (cm) and crocin
content (%). Material under study for identification of high yielding clones comprised
of 500 corm samples of uniform corm weight (>10 g) collected from saffron growing
area of Kashmir during the month of August-September. Each sample was based on
30 corms which were planted as single observational rows at spacing of 20 x 10 cm
and were replicated twice in RBD design. Observations were recorded on various
floral, yield and quality traits over 3 years. Standard procedures were used to compute
analysis of variance and components of variance (Verma et al., 1987; Johnson et al.,
1955).
Induction of Genetic Variability through Mutagenesis
4 sets of saffron corms with each set containing 200 corms of uniform corm
weight (>10.0 g) were irradiated with 0.25, 0.50, 0.75 and 1.0 Krad doses of Co 60
gamma rays at BARC, Srinagar. A set of 200 untreated corms was used as control.
Irradiated corms were planted with plant geometry of 20 x 10 cm on raised buds.
Observations were recorded on various morphological and yield related traits.
Identified mutants were advanced to M2 generation for further study
In-vitro Micropropagation
Hardened in vitro corms developed at Regional Research Laboratory, Jammu
through somatic embryogenesis, auxillary shoot formation and direct organogenesis
were evaluated under temperate conditions of Kashmir Valley for average corm
weight (g), germination (%), average number of radical leaves, average weight of
corms (g) after one year and gain in corm weight (g). Corms were categorized in 4
groups based on initial corm weight.
RESULTS AND DISCUSSION
Extent of Variability and Identification of Elite Clones
Evaluation of 438 flower samples collected from temporal sub populations of
Jammu & Kashmir revealed a wide range of variability for floral, yield and quality
attributes. Flowers completely devoid of style and anthers and freaks with 4 and 5
stigmas were observed from the natural population, however, such plants did not
inherit the presence of supernumerary stigmas to their progenies .The presence of
higher number of stigmas per flower results from physiological and/or developmental
irregularities leading to 4-5 stigmas. However, detection of flowers with increased
number of stigmas in natural population is a universal phenomenon (Estilai, 1978;
Dhar et al., 1988). Magnitude of variability (Table 1) revealed that the mean fresh and
dry weight of 100 flowers in the temporal sub-population of Jammu & Kashmir was
23.46 and 3.82 g, respectively, yielding approximately 0.759 g of laccha grade saffron
(Pistil).Thus, 1Kg fresh flowers produce approximately 32 g of dried laccha saffron.
Pistil weight ranged from 1.76-5.88 g on fresh weight basis revealing possibility of
saffron improvement through selection. Similar results of wide range of variability in
Kashmir have also been reported (Zargar, 2002). Stigma length ranged from 2.41-3.87
cm with a mean value of 2.5 cm, whereas, Crocin content (%) ranged from 9.92-14.35
percent with a mean value of 12.0 percent. Quality evaluation confirmed that saffron
of Kashmir is of intrinsically high quality with respect to colouring pigment and
traditional post harvest processing was responsible for poor saffron quality. The
results are in general agreement with earlier reports (Nehvi et al., 2005). Evaluation of
500 saffron genotypes collected from natural population revealed wide range of
variability for different traits. no. of flowers per spathe ranged from o.65-5.58 with
fresh flower weight ranging from 172–355 mg. Economic product (Pistil weight) on
fresh weight basis ranged from 14.37-68.42 mg, whereas, on dry weight basis it
ranged from 6.00-14.60 mg. Stigma length which contributes to the yield potential
varied from 1.75 cm to 3.72 cm with a corresponding variability for style length
ranging from 1.70 cm – 4.25 cm. Average number of daughter corms ranged from
2.37–7.05, whereas, average weight ranged from 1.59-8.49 g. Colouring pigment
(crocin content) ranged from 8.55-17.10 percent. Greater magnitude of phenotypic
variance than the corresponding genotypic variance was observed which indicated
greater influence of environment in the inheritance of floral, corm and quality
attributes (Table 2). Heritability in the broad sense was recorded low for all traits,
excepting fresh flower weight, fresh pistil weight and stigma length which revealed
high broad sense heritability. Genetic gain as percent of mean was high for number of
flowers/spathe, fresh flower weight, fresh pistil weight and Crocin content, whereas, it
was medium for dry pistil weight, stigma length and number of daughter corms and
low for rest of the traits. Components of variability indicate ample scope of saffron
Improvement through selection among the temporal sub population of J&K. Grilli
Caiola et al., 2001 also reported phenotypic differences in respect of flower size, tepal
shape and colour intensity.
Evaluation of 500 saffron genotype for yield and yield related traits over 3
years lead to the identification of 10 superior genotypes viz; SMD-3, SMD-11, SMD31, SMD-45, SMD-52, SMD-68, SMD-79, SMD-81, SMD-21 and SMD-224. Elite
genotypes recorded saffron yield ranging from 4.0-7.6 kg/ha with a corresponding
crocin content ranging from 13.89-17.10 percent. Superior performance of 3
accessions in Iran has also been reported (Parviz et al., 2004). In Kashmir elite subpopulation developed from the progenies of the corms selected from extensive saffron
belts have been identified and catalogued (Munshi and Zargar, 1991). With the
commercial cultivation of elite genotypes the productivity level of Jammu & Kashmir
is expected to enhance.
Induction of Genetic Variability through Mutagenesis
Mutagenesis is another approach which was applied to induce genetic
enhancement for yield attributing traits. Existing saffron population was irradiated
with gamma rays from a Co60 source at 0.25, 0.50, 0.75 and 1 Kr dose. Irradiation
doses had significant effect on plant height, Sprouting, Survival %, number of
flowering plants, number of daughter corms, corm yield, number of flowers and pistil
recovery/100 pistils (Table 3). Induction of early sprouting in 0.25 Kr and 0.5 Kr and
delayed sprouting in higher doses was noted. Radiation at lower doses (0.25 Kr)
enhanced plant biomass, whereas, a reverse reaction was observed at higher doses.
M1 mutants with 0.25 Kr radiation dose showed enhanced corm yield and saffron
yield by a margin of 53.44 % and 84.28 %, respectively. Increased saffron recovery
has resulted from more number of flowers/plant. Plant survival was reduced to 79 %
at 1.0 Kr. Radiation treatment at higher doses did not reveal any significant effect on
number of daughter corms, corm yield, number of flowers and saffron yield. The
results are in general agreement with other reports (Akhund-Zade and Mazaferova,
1975; Laneri et al., 1983; Khan, 2004). Fifty superior mutants from M1 generation
with distinct yield superiority were identified and were advances to M2 generation. In
M2 generation superiority of mutants in terms of saffron recovery was maintained to
the tune of 47.36 percent (Table 4). Elite mutants also revealed superiority in terms of
pistil length, increased number of flowers/spathe.
Delayed sprouting, slow growth in higher doses, increase/decrease plant
height at lower and higher doses, respectively and induction of flowering in M1
generation at lower doses, are due to auxin synthesis, whereas, at higher doses, it is
inhibited resulting into delaying, and Stunted growth. The present study is in
confirmation with other reports (Gordon, 1959). Stunted growth, reduction in survival
and reduced fertility is also attributed to genetic loss due to chromosomal aberrations
and gene mutations (Datta and Gupta, 1980). Mutations in the biochemical pathways
assist, the synthesis of flower inducing substances, leading to formation of flower.
In Vitro Micropropagation
Since the saffron reproduce only vegetatively by the corms nay attempt to
modernize saffron cultivation will therefore require efficient mass production of
pathogen free corms. For mass propagation of corms tissue culture is the only
effective alternative (George et al., 1992). Tissue cultured studies have shown
possibility to regenerate saffron plantlets and in vitro corm development through
Somatic embryogenesis, auxiliary short formation and direct organogenesis.
Development of tissue cultured cormels at RRL, Jammu was asynchronous and corms
of different size and shape were produced. 201 corms produced through Somatic
embryogenesis averagely weighed from 0.04-2.73 g. For field evaluation of tissuecultured corms, 4 sub-groups were made based on the average weight of Corms.
Majority of the in-vitro corms (165) weighed less than 1g, whereas, 22 corms
weighed from 1-1.5g. An average weight of 1.5-2.0 g was exhibited by 9 corms and
only 5 corms showed an average corm weight above 2 g. The hardened tissue cultured
cormels were planted for field evaluation.
Field evaluation studies of in vitro corms revealed importance of corm weight.
Survival percentage among the corms having average corm weight less than 1g was
only 26 %, whereas, it was 83.66 % among the corms having average corm weight
above 1 g. Maximum number of radicle leaves was observed among the corms having
average corm weight above 2.0 g. Corms having average corm weight between 1.01
to 1.96 g behaved equally with regard to average number of radicle leaves. However,
corms having average corm weight 1.58 to 1.96 were superior in length of radicle
leaves. Corm development of tissue cultured corms was directly proportional to initial
corm weight. Average corm weight after one year of plantation ranged from 0.80
(Group 4) to 5.97 g (Group 5) showing a weight gain ranging from 0.25 g (Group 3)
to 3.54 g (Group 5). For corms weighing 1.58 to 1.96 g the average corm weight on
uprooting was 3.22, however, with further decline in initial weight the corm
development was slow (Table 5). The protocol needs to be revised in view of corm
size so as to make it commercially viable and acceptable.
ACKNOWLEDGEMENTS
The authors acknowledge the financial support and guidance provided by the
Indian council of Agricultural research, National Horticulture Board (Government of
India) and SKUAST-K.
Literature Cited
Akhund-Zade. and Muzaferova, R.S. 1975. Studies of the effectiveness of gamma
irradiation of the saffron. Radiobiologiya. 15(2):319-322.
Ahuja, A., Koul, S., Ram G. and Kaul, B.L. 1994. Somatic embryogenesis and
regeneration of plantlets in saffron. Ind.J.Exptl. Biol. 32:135-140.
Chichirricco,G.1990. Sterlity and improvement of saffron Crocus:In Tammaro and
Marra (eds): Lo Zafferano: Proceeding of the International Conference in saffron
(Crocus sativa L.) L” Aquila (Italy) 27-29 October 1989. Universita Degli Studi
L’ Aquila e Academia Italiana della Cuema, L’Aquila.
Datta, S.K. and Gupta, M.M.1980. Effect of gamma irradiation on rooted cuttings of
small flowered chrysanthemum, New Bot. VII:7 3-85.
Dhar, A.H., Sapru, R. and Rebha. K.1988. Studies on saffron in Kashmir. Variation in
natural population on cytological behaviour. Crop Improv. 15(1): 48-52.
Estilai, A. 1978. Variability in saffron (Crocus sativus L.). Experientia 34:725-727.
Gordon, S. A.1957. The effect of ionizing radiation on plants: Biochemical and
physiological aspects, Quart. Rev. Biol. 32:3-14.
George, P.S., Visavanath, S., Ravishankar, G.A. and Venkataraman L.V. 1992. Tissue
culture of saffron: Somatic embryogenesis and shoot regeneration. Food Biotech.
6:217-223.
Grilli Caiola, M., Di Somma, D. and Lauretti, P.2001. Comparative study on pollen
and pistil of Crocus sativus L. (Iridaceae) and its allies species. Ann. Bot. (Roma)
1:93-103.
Khan, I.A. 2004. Induced mutagenic variability in saffron (Crocus sativus L.) Acta
Hort. 650:281-283.
Johnson, H.W., Robinson, H.F. and Constock, R.E. 1995. Estimates of genetic and
environmental variability in soyabean. Agron. J. 47:314-318.
Laneri, U. and Lucretti, S. 1983. Propagazione in vitro di Crocus sativus L. rapporto
dei resultati ottenuti nel 1982. ENEA. Rome, RT/FARE-SIN (83).
Munshi, A.M. and Zargar, G.H. 1991. Variation in the natural population of saffron
(Crocus sativus L.) crop in Kashmir and performance of some selected subpopulations. Phytobreedon. 71 (1 & 2):62-67
Nehvi F.A., Agarwal, S.G., Mir, M.A., Dar, S.A., Mir, Z.A. and Nusrat
Nabi.2005.Quality drying of Saffron SKUAST J Res. 7(2):343-346
Parviz, E., Yadollahi. A. and Maibodi, M. 2004. Productivity, growth and quality
attributes of 10 Iranian saffron accessions under climatic conditions of ChahaMahal Bakhtiari central Iran. Acta Hort. 650:183-88.
Verma, M.M., Gill, K.S. and Vivk, D.S. 1987. Genotype-Environment interaction, its
measurement and significance in plant Breeding. Communication Centre Punjab
Agricultural University Ludhiana.
Zargar, G.H. 2002. Genetic variation in saffron and importance of quality seed corms.
National Seminar Cum Workshop Srinagar. August, 2002.
Table 1. Nature and magnitude of phenotypic variability for flower and its economic
components in the temporal sub-population of saffron crop in J&K.
Characters
Mean
Components of variability
Range
CV %
Fresh flower wt (g)
23.46 ± 3.31
15.86-36.69
14.14
Fresh Perianth wt (g)
16.73
±
2.15
11.03-28.22
12.89
Fresh Anther wt (g)
3.21
±
0.62
1.43-4.90
19.31
Fresh Pistil w t(g)
3.52
±
0.47
1.76-5.88
13.42
Dry flower wt (g)
3.82
±
0.71
1.41-6.41
18.78
Dry Perianth wt (g)
2.37
±
0.34
1.41-3.58
14.66
Dry Anther wt (g)
0.692
±
0.11
0.230-1.61
16.51
Dry Pistil wt (g)
0.759
±
0.13
0.39-1.59
18.32
Stigma length (cm)
2.5
±
0.46
2.41-3.87
18.49
Style length (cm)
2.0
±
0.56
1.50-3.10
28.20
Pistil length (cm)
4.5
±
0.69
3.90-6.70
15.44
Crocin %
12.01
±
1.25
9.92-14.35
10.44
Table 2. Components of variability for different floral, morphological, corm and
quality attributes in saffron genotypes
Components No. of Fresh
Fresh
Dry
Stigm Style No. of
Avg. Crocin
flower flower
pistil
Pistil
a
lengt daughter corm
(%)
/
wt(mg)
wt
wt
length
h
corms
wt.(g)
spathe
(mg)
(mg)
(cm) (cm)
1
2
3
4
5
6
7
8
9
10
Range
0.65 –
172 14.376.0 1.75- 1.702.371598.555.58
355
68.42 14.60
3.72
4.25
7.05
8.49
17.10
62 g
0.218 1924.2 16.60
0.85
0.053 0.05
0.66
0.57
4.64
7
62 p
0.937 2984.0
20.7
2.56
0.085 0.55
2.85
6.9
6.52
1
GCV
32.24
17.02
19.91
14.9
10.35 7.14
19.65
12.66
17.47
PCV
67.18
21.2
22.24
25.9
13.22 16.0
38.71
44.06
20.70
2
h2
0.23
0.65
0.80
0.33
0.61
0.20
0.23
0.08
0.712
G.A
0.47
72.56
7.52
1.09
0.37
0.30
0.81
0.45
3.75
G. gain (%
32.21
28.16
36.78
17.6
16.70 6.55
18.48
7.46
30.4
of mean)
Table 3. Effect of radiation doses on saffron development in M1 generation
Characters
Control
0.25 krd
0.5 krd
0.75 krd
Plant height(cm)
21.00 + 1.30
43.00 + 3.10
39.0 + 2.8
19.5 + 1.9
Days to sprouting
50+1.90
45 + 1.80
46 + 2.00
60 + 1.400
Survival Plants
97.00
99.00
98.00
86.5
(%)
Flowering plants
98.0
100.00
89.00
81.00
(%)
No. of daughter
1.9 + 0.6
256 + 1.50
2.42 + 2.0
2.16 + 2.9
corms/plant
Corm yield/plant
5.80 + 1.4
8.96 + 1.80
7.05 + 1.9
6.84 + 1.9
(g)
No. of
1.46 + 0.30
1.87 + 0.20
1.55 + 0.25
1.54 + 0.30
flowers/plant
Fresh pistil wt
4.200 + 1.500
5.790 + 1.30 4.800 + 0.790
4.750 +
(100 pistils) g
1.00
Dry pistil
0.700 + 0.200
1.290 + 0.320 1.00 + 0.100 1.00 + 0.100
(wt/100 pistils) g
1.0 krd
22.00 + 1.9
61 + 2.00
79.00
72.00
1.98 + 1.5
5.65 + 1.4
1.44 + 0.15
4.180 + 0.900
0.680 + 0.250
Table 4. Performance of mutants in M2 generation
Population Plant Days to
No. of
Fresh
height
sprout flowering pistil wt.
(cm)
plants
(100
(%)
pistils)
(g)
Natural
population
Mutant
population
35.0
45.00
0.97
3.900
Dry
pistil
wt.
(100
pistil)
(g)
0.950
49.0
47.00
2.15
8.670
1.400
Stigma
length
(cm)
Style
length
(cm)
Pistil
length
(cm)
2.950
3.220
6.170
3.110
13.520
6.630
Table 5. Performance of hardened in vitro corms under field conditions of Kashmir
No. of Total Initial
Range
Germination
Avg.
Avg.
After 1 year
corms corm
avg.
(%)
No. of
Length of
Avg.
Grain
wt. (g) corm
radicle
radical
Wt. of
in
wt. (g)
leaves
leaves
corms weight
(cm)
(g)
(g)
5
12.13
2.43
2.13-2.73
60.0
5.0
22.17
5.97
3.54
9
16.45
1.83
1.58-1.96
100.0
3.78
25.83
3.22
1.45
22
26.12
1.19
1.47-1.61
91.0
3.40
23.78
2.19
0.25
165
67.16
0.41
0.04-0.88
26.0
2.23
18.13
0.80
0.34
201 121.86 1.46
0.04-273
69.25
3.60
22.47
3.04
1.39