International Journal of Agricultural

Science and Research (IJASR)

ISSN(P): 2250-0057; ISSN(E): 2321-0087
Vol. 8, Issue 3 Jun 2018, 147-152
© TJPRC Pvt. Ltd.

MICROSPORE CULTURE EFFECTS ON DOUBLE HAPLOID PRODUCTION

IN LENTIL (LENS CULINARIS MEDIK)

KAPIL DESWAL & VIJAIPANDURANGAM

Department of Plant Physiology,
Institute of Agricultural sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
ABSTRACT

Establishment of an effective and reproducible protocol is one of the basic prerequisites for the improvement of
any crop. Double-Haploid technology has proven to be breakthrough in many crop species and results in the production
of homozygous plants in a single step. In spite of a number of success reports of plant tissue culture, technology, very few
satisfying and reproducible protocols were available in legumes like a lentil. This study, carried out forthe production of
DH plants in the different medium, with various hormone treatments in order to establish a reproducible protocol for
microspore regeneration of the cultivar HUL-57. For this determination, the effects of 2 different mediums with 2
hormones on microspore of lentil either through callus induction or directly regeneration were studied. The experiment
was done with LS and N6 medium in presence of BAP and 2,4-D results in the non-significant induction of callus from

Original Article
microspores. From the current experiment, this is found that either of the media in various hormones (BAP and 2,4-D)
concentrations are non-responsive and became difficult even after combination of different stresses like cold and
centrifugation. These reports of non-responsive behavior will be pointing towards trying different medium with the same
objective to get the completely homozygous plants.

KEYWORDS: Callus, Hormones, Regeneration & Lentil (Lens culinarisMedik)

ABBREVIATIONS: DH – Double Haploid; BAP – N6-benzylaminopurine; 2,4-D – 2 & 4-dichlorophenoxyacetic Acid

Received: May 01, 2017; Accepted: May 22, 2018; Published: Jun 18, 2018; Paper Id.: IJASRJUN201816

INTRODUCTION

Lentil (Lens culinarisMedik.) is an important pulse crop and its production takes place in cool season
which grown all over the world. Lentil crop seeds have very much nutritive value, especially in developing
countries and, their protein content is 20–36% more compared with 8–12% for cereals (Christou, 1993. For plant
breeding and molecular genetics programs, double haploid are very important tools. Haploids and DH can be
produced by various methods such as apogamy, wide crosses and androgenesis. Conventional breeding methods are
not only time consuming, but also not feasible because fully homozygosity is not retained in haploid produced.
On the other hand, the recalcitrant nature of legumes species towards DH production is also a great problem.
However, regeneration of haploid through induction of androgenesis in some legume species was reported.

To produce embryos through the androgenesis pathway, proper induction of cell division and
differentiation is modulated by many factors like, genotype, growth condition of donor, pre-treatment of flower
buds, and stages of microspore culture. Along with that, stress pre-treatment also important for proper induction like
centrifugation treatment, cold treatment, and osmotic shocks shown a positive effect on induction of haploids.
Within legume species, several haploid plants from isolated microspore have been reported in few species like

www.tjprc.org [email protected]
148 Kapil Deswal & Vijaipandurangam

pigeon pea (Cajanuscajan L.) (Kaur and Bhalla 1998), field pea (Pisumsativum L.) and grasspea (Lathyussativus
L.)(Ochattet al. 2009) and more recently in chickpea (Cicerarietinum L.)( Grewelet al. 2009).

A cold temperature pre-treatment of the immature flower buds prior to microspore isolation was reported
important in microspore embryogenesis (Kaur and Bhalla 1998), and recently specific cold pre-treatment at 4oC for 72
hours enhanced induction of androgenesis in chickpea anthers (Grewalet al.2009). Centrifugation as an extra stress
treatment for induction of androgenesis in legume species which showed beneficial effects in chickpea (Altaf and Ahmad
1986 ; Grewalet al. 2009), and in lupin microspores (Campos-Andradaet al., 2001; Baylisset al., 2004).

Keeping in view of the above facts, the aim of the present study is to develop an efficient protocol for successful
induction of haploid from microspore culture of lentil. Lentil is the least exploited species in term of DH production.
Keller and Ferrie, (2002) reported calli induction, but not able to regenerate plantlets from that call us. In another study by
Croser and Lulsdorf (2004) reports shows that microspores of CDC Crimson and CDC Robin not able to regenerate
embryos.

The final objective of this research was to identify the effects of different medium, in various hormone
concentrations to produce double-haploid from isolated microspores of lentil.

MATERIALS AND METHODS

The experiment was conducted during the rabi season 2016-2017to find strategies for developing Double-Haploid
of lentil through microspore culture techniques in different culture media in variety of hormone combinations at the
laboratory of institute of agricultural sciences, Banaras Hindu University. The experimental site is located at is situated
25015’ North Latitude and 60003’ East Longitude with an altitude of 128.93 m above sea level.

Plant Material and Growth Conditions

The lentil cultivar HUL-57 was used to experiment. The lentil variety was grown in pots at the farmhouse of
institute of agricultural sciences, Banaras Hindu University. Mature seeds were washed using Tween80 (0.5mL/L) for 5
minutes before sterilization. Seeds were surface-sterilized by immersion in 70 % ethanol for 5 minutes, followed by 3
rinses in sterile distilled water. Lentil seeds are grown in plastic pots under favorable conditions. During the vegetative
growth of crop plants, adequate supply of water and nutrients are given to avoid any stress which halts plant growth and
development. After successful completion of vegetative phase, crop transitions to reproductive phase.

Donor Plants, Genotype, Bud Size and Microspore Stage

Donor plants grown in a favorable environment, without any stress, are a necessity for an androgenic response.
Bud size and microspore stage are also important and mainly uninucleate microspores with their high auxin content are
used for androgenesis in lentil.

Low Temperature Stress Treatment

Lower temperatures not only increase the length of testing time, but also helpful in obtaining callus and
heart-shaped-stage embryos. Buds were directly isolated and pre-treated at 40C for 72 hours. After the stress, buds put in a
100ml beaker and surface sterilized by stirring in 70 % ethanol for 5 minutes. Buds are transferred to a sterilize mother and
microspores were released by squashing buds with a pestle. The resulting microspores were passed through a cheesecloth

Impact Factor (JCC): 6.1964 NAAS Rating: 4.13

Microspore Culture Effects on Double Haploid Production 149
in Lentil (Lens Culinaris Medik)

filter. Microspore were transferred to centrifuge tube which contains 2,4-D solution ( 0.1 mg/l). Centrifuge for 3 minutes at
5000 RPM. After that, the supernatant is discarded and microspore was used for inoculation in N6 and LS medium.

Culture Media

Two types of culture medium were used (CHU (N6) (Table 1) and LS (Table 2)) for double haploid production
and their Culture medium composition with different hormones (BAP and 2,4-D) summarized (Table 3).

Hormone Preparation

BAP and 2,4-D hormones are used in three different combinations with the medium LS and N6.
The hormone concentrations used in the experiment are 0.1 mg/l and 0.5 mg/l in different combination with both the
medium which are tabulated in table 3. Each combination is replicated three times with both the medium used.

Table 1: Growth Regulator Composition of Culture

Media used in Callus Induction and Regeneration
Medium Hormone (mg/1)
A1 0.5 2,4-D + 0.5 BAP
A2 0.5 2,4-D + 0.1 BAP
A3 0.5 BAP + 0.1 2,4-D
B1 0.5 2,4-D + 0.5 BAP
B2 0.5 2,4-D + 0.1 BAP
B3 0.5 BAP + 0.1 2,4-D

Two types of media used, A1, A2 and A3 contains LS medium and

B1, B2, and B3 contains CHU (N6) medium with (Bavistin and Amphicllin)

(30mg/l)) 3% (w/v) sucrose and 0.8% (w/v) agar.

RESULTS AND CONCLUSIONS

Culture Medium and Callus Induction

Lentil microspores were cultured on CHU (N6) and LS media with various concentrations of 2, 4-D and BAP
hormones are used for production of Double-haploid but the result shows that there was no significant difference with
respect to the effect of various hormonal treatments and different types of media. In some of the treatments, it seems to
form some callus like projections, but the results are not significant with either of the media used for DH induction.
However, 2,4-D hormone was shows responses when used with a modified MS medium with B5 vitamins and a two-fold
concentration of CaCl2 for culturing lentil explants Bagheriet al. (2012).

The earlier data work reports in lentil from Keller and Ferrie, (2002) and Croser and Lulsdorf, (2004) showed that
through microspore culture, technology, it is not possible to regenerate either Haploid or diploid plantlets. And if some
induction reported up to callus levels, finding problems in determining whether the induced calli originate from
gametophytic or sporophytic tissue. The absence of a robust haploid production system for and rogenesis could motivate us
to finding a useful protocol through suitable culture conditions. Without the valid protocol for a particular species,
the current experimental efforts to adapt Double Haploid production techniques to recalcitrant species like lentil will
continue to be difficult and time consuming.

www.tjprc.org [email protected]
150 Kapil Deswal & Vijaipandurangam

However, even under the finest situations, plant regeneration through LS and CHU (N6) medium remains difficult
even after combination of different stresses like cold and centrifugation. From the current experiment, this is finding out
that either of the media in various hormones (BAP and 2, 4-D) concentrations is non-responsive. In future, it opens the
research approaches in different directions, this analysis could influence the choice of different media composition or same
medium with different hormone stimulators in future. Alter the design of the experiment enhances the chances of getting a
fruitful result. Efforts are in progress to adapt this technology to lentil to improving efficiency and integrating these
techniques into routine breeding programs to accelerate genetic or breeding productivity goals.

Table 2: Linsmaier and Skoog Media (LS) Compostion

Ingredients mg/l
Ammonium nitrate 1650.000
Calcium chloride 332.200
Magnesium sulphate 180.690
Potassium nitrate 1900.000
Potassium phosphate monobasic 170.000
Boric acid 6.200
Cobalt chloride hexahydrate 0.025
Copper sulphate pentahydrate 0.025
EDTA disodium salt dehydrate 37.300
Ferrous sulphate heptahydrate 27.800
Manganese sulphate monohydrate 16.900
Molybdic acid (sodium salt) 0.213
Potassium Iodide 0.830
Zinc sulphate heptahydrate 8.600
myo-Inositol 100.000
Thiamine hydrochloride 0.400
Sucrose 30000.000
Bavistin 30
Amphiclin 30

Table 3: CHU (N6) Media Composition

Ingredients mg/l
Ammonium sulphate 463.000
Calcium chloride 125.340
Magnesium sulphate 90.370
Potassium nitrate 2830.000
Potassium phosphate monobasic 400.000
Boric acid 1.600
Ferrous sulphate heptahydrate 37.300
Manganese sulphate monohydrate 27.800
Potassium Iodide 3.330
EDTA disodium salt dihydrate 0.800
Zinc sulphate heptahydrate 1.500
Nicotinic acid (free acid) 0.500
Pyridoxine HCl 0.500
Thiamine hydrochloride 1.000
Glycine 2.000
Bavistin 30
Ampicillin 30

Impact Factor (JCC): 6.1964 NAAS Rating: 4.13

Microspore Culture Effects on Double Haploid Production 151
in Lentil (Lens Culinaris Medik)

REFERENCES

1. Bagheri1*, V. Ghasemi Omraan2 and S. Hatefi3, (2012), Indirect in vitro regeneration of lentil (Lens culinarisMedik.), Journal
of Plant Molecular Breeding (JPMB) Vol.1/No.1, pp. 43-50.

2. M. R. Ferrie and K. L. Caswell, (2011) “Isolated Microspore Culture Techniques and Recent Progress for Haploid and
Doubled Haploid Plant Production,” Plant Cell, Tissue, and Organ Culture, Vol. 104, No. 3, pp. 301-309.

3. Altaf, N. and Ahmad M. S. (1986) Plant regeneration and propagation of chickpea (Cicerarietinum L.) through tissue culture
techniques. In: I. A. E. Agency (ed.) Nuclear Techniques and in vitro Culture for Plant Improvement. Vienna, Austria, pp. 407–
417.

4. Bayliss, K. L., Wroth, J. M. and Cowling, W. A. (2004) Pro-embryos of Lupinus spp. produced from isolated microspore
culture. Australian Journal of Agricultural Research 55, 589–593.

5. Campos-Andrada, da Paz M., Filomena-Carneiro, M. and Mota M., (2001) Isolated microspore culture in Lupinusmutabilis
Sweet. AgronomiaLusitana 49, 119–135.

6. Christou, P. 1993. Biotechnology of crop legumes. Euphytica, 74:165-185

7. Croser, J. S. and Lulsdorf, M. M. (2004) Progress towards haploid division in chickpea (Cicerarietinum L.), field pea
(Pisumsativum L.) and lentil (Lens culinarisMedik.) using isolated microspore culture. In: European Grain Legume
Conference, AEP, Dijon, France, pp. 189.

8. David J. Williams And Alan Mchughen, (1986) Plant regeneration of the legume Lens culinarisMedik. (lentil) in vitro, Plant Cell,
ltssue and Organ Culture vol., PP. 7149-153

9. Grewal, R. K., Lulsdorf, M., Croser, J., Ochatt, S., Vandenberg, A. and Warkentin, T. D. (2009) Doubledhaploid production in
chickpea (Cicerarietinum L.): role of stress treatments. Plant Cell Reports 28, 1289–1299.

10. Iva Smýkalová1, Martina Větrovcová1, Miroslav Klíma2, Ivana Macháčková3 And Miroslav Griga1, (2006) Efficiency of
Microspore Culture for Doubled Haploid Production in the Breeding Project “Czech Winter Rape”, Czech J. Genet. Plant
Breed., VOL.42, PP. 58–71.

11. Kaur, P. and Bhalla, J. K. (1998) Regeneration of haploid plants from microspore culture of pigeonpea (Cajanuscajan L.).
Indian Journal of Experimental Biology 36, 736–738.

12. Keller, W. A. and Ferrie, A. M. R. (2002) Development of haploid production technology applicable for genetic improvement
of lentils. ADF project No. 98000297.

13. Lulsdorf, Monika &Croser, Janine &Ochatt, Sergio. (2011). Androgenesis and doubled-haploid production in food legumes.
Biology and Breeding of Food Legumes. 159-177.

14. Ochatt, S. J., Pech, C., Grewal, R., Conreux, C., Lulsdorf, M. M. and Jacas, L. (2009) Abiotic stress enhances androgenesis
from isolated microspores of some legume species (Fabaceae). Journal of Plant Physiology 166, 1314–1328.

15. R. Khound, M. Santra, P. Baenziger and D. Santra, (2013) "Effect of Cold-Mediated Pretreatment on Microspore Culture in
Winter and Spring Wheat," American Journal of Plant Sciences, Vol. 4 No. 11, pp. 2259-2264.

16. SaeidHassanzadehMobini& Monika Lulsdorf& Thomas D. Warkentin& Albert Vandenberg (2015) Plant growth regulators
improvein vitro flowering and rapid generation advancement in lentil and faba bean, In Vitro Cell.Dev.Biol.—Plant VOL. 51,
PP. 71–79.

www.tjprc.org [email protected]