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Production of haploids of cardamom (Elettaria cardamomum Maton.) through anther culture / microspore
culture
(1998-2002) Final report
Submitted to Indian Council of Agricultural Research
INDIAN INSTITUTE OF SPICES RESEARCH (Indian Council of Agricultural Research) Marikunnu P.O., Calicut � 673 012, Kerala
Contents Page
Annual report proforma 1
Objectives 1
Budget 2
Progress of Research (Annexure 1) 5
Introduction 6
Review of Literature 8
Materials and Methods 15
Results and Discussion 19
Summary and conclusions 39
References 41
Detailed Expenditure Statement (Annexure II): 48
Comments of Project Co-ordinator/Referee 52
(Annexure III)
FINAL REPORT FOR RESEARCH SCHEME
1. Project Title : Production of haploids of cardamom (Elettaria cardamomum Maton.) through anther culture / microspore culture.
2. Sanction No. : F. No. 15(17)/95 � Hort. I Dated 24th July1997.
3. Date of start : 1-4-1998
4. Date of Termination : 31-3-2002
5. Institutions name : Indian Institute of Spices Research
Place : Marikunnu
District : Calicut
State : Kerala
Dept./Div. Name : Division of Crop Improvement and Biotechnology
Actual Location : Indian Institute of Spices Research, Calicut (Location of research to be carried out)
6. Principal Investigator
Name : Dr. PN Ravindran ( PC Spices till March 2000)
Dr. K. Nirmal Babu ( After March 2000)
Designation : Sr. Scientist
Div./Section : Crop Improvement and Biotechnology
Address : Indian Institute of Spices Research Marikunnu P.O. Calicut, Kerala E mail : [email protected]
7. Objectives
! The anther / microspore culture technology and subsequent production of dihaploids is an important means by which homozygous lines could be achieved for the subsequent production of high yielding hybrids exhibiting maximum heterosis.
! Production of haploids through the ' microspore callus ' is a sure way of introducing variation in the crop. The variations obtained may be useful in breeding, especially for developing disease tolerant lines.
! In cardamom, resistance to the katte virus seems to be a recessive character or a character controlled by cytoplasmic factors. Through anther / microspore culture, it is possible to fix the recessive genes in homozygous condition.
! Cardamom is a naturally cross-pollinated crop and the dihaploids from such hybrid plants are recombinant homozygous products useful in the fixation of gene loci. Additive effects are fixed in dihaploids.
! The ultimate aim of the project is to evolve high yielding disease resistant cardamom lines through crossing of dihaploids.
9. Duration of Scheme : 04 years. - 00 Months - 00 Days
10. Total Cost of Scheme : Rs. 7,78,430.00
Recurring Recurring (Contingency + TA+ Institutional charges) : Rs. 2,48,139.00
Pay of Officers : Rs. 4,13,035.00 Rs. 6,61,174.00 Name of Post Pay Scale No. of Post Total Scientist Junior Research Fellow Senior Research Fellow
- - -
- - -
- - -
Research Associate Others
8800.00 + 1320.00 (HRA) Nil
1 1,21,440.00
Year
Pay of officers Pay of Establishment
TA Other Allow-ances
PF Contingency Instt. charges
Total
I 1,21,440.00 - 10,000.00 - - 1,00,000.00 16,220.00 2,47,660.00
II 1,21,440.00 10,000.00 - - 1,00,000.00 16,370.00 2,47,810.00
II
1,21,440.00
10,000.00
- - 1,00,000.00 16,520.00 2,47,960.00
Total 3,64,320.00 30,000 - - 3,00,000.00 49,110.00 7,43,430.00
B. Non-Recurring 35,000.00
Total Budget Year Recurring Non-recurring Total I 2,47,660.00 35,000.00 2,82,660.00 II 2,47,810.00 2,47,810.00 III 2, 47,960.00 2,47,960.00 IV (Extension period)* 7,43,430.00 35,000.00 7,78,430.00 * No separate sanction for the extension period, The balance amount in the end of third year was sanctioned. 11. Total Amount sanctioned : 7,78,430.00
12. Total Amount Spent : 7,23,430.00
(as on 31.07.2002)
See Annexure-II for details Consolidated statement of expenditure (1998�2002) Year Amount
Sanctioned Opening balance
Amount released
Amount spent
Balance Balance provision@
1998�1999 2,82,660 - 2,13,420 1,85,939 27,481 -1999�2000 2,47,810 27,481 2,85,370 2,33,791 79,060 -2000�2001 2,47,960 79,060 1,16,060 97,457 97,663 -2001-2002 - 97,663 1,08,580 1,41,700 64,543 -*2002 May - **64,543 - **64,543 0 -Total 7,78,430 - 7,23,430 7,23,430 0 55,000
** The expenditure committed in March, 2002, and the payments made in May 2002 @ The amount under balance provision need not be released.
13. Result of Practical / Scientific value:
♣ Production of callus from cardamom anthers.
♣ Regeneration of shoots from cardamom anthers
♣ Rooting of shoots and anther derived plants were established in hardening facility.
14. Papers Published : Nil
Manuscripts submitted : Nil
Papers presented at scientific meetings : Nil
Manuscripts under preparation : Nil
15. Detailed Progress Report : See Annexure - I Signature Principal Investigator Name : Dr. K. NIRMAL BABU Designation : Senior Scientist Indian Institute of Spices Research Marikunnu P.O. Calicut, Kerala.
Director or Head of Institution / Station Date: 16. Comments of the Project Co-ordinator / Referee : See Annexure - III 17. Remarks of the Council:
ANNEXURE - I
Production of haploids of cardamom (Elettaria cardamomum
Maton.) through anther culture / microspore culture
ANNEXURE - I Progress of research Appointments
The project was started with the joining of the research associate on 15th April
1998, at Indian Institute of Spices Research, Calicut.
Dominic Joseph Research Associate 15.04.1998 � 31.08.2000
Benny Daniel Research Associate 30.03.2001 - 30.09.2001
Tajo Abraham Research Associate 06.10.2001 � 19.11.2001
Technical programmes
1. Standardization of optimum age of panicle and anther suitable for culture.
2. Standardization of cold treatment procedure, sterilization and inoculation procedure
3. Standardization of photoperiod and light conditions
4. Studies on pollen callus and pollen embryo development
5. Standardization of plant regeneration medium
6. Enhancing the repeatability of plant regeneration from anthers and anther derived
callus.
7. Standardization of rooting and hardening
8. Cytological indexing of anther/anther callus derived plants and identification of
haploids.
9. Microspore culture for enhanced haploid production.
Introduction
Elettaria cardamomum Maton, cardamom, also known as the queen of spices, is a
native of the evergreen forests of South India. A perennial rhizomatous plant belonging to
the Zingiberaceae family, is cultivated widely for its fruit, a capsule, which when mature
and dry yields the cardamom of commerce. Cardamom is an important spices valued since
time immemorial, for its pleasant flavour and is used directly for domestic and culinary
purposes. Guatemala and India are the major cardamom producing centers and India
earned a foreign exchange of Rs. 2760.3 lakhs by exporting 550 tonnes of cardamom in
1999-2000. About 7000 metrictones of cardamom are consumed in India itself every year.
The Indian Institute of Spices Research, (IISR) holds more than 300 accessions of
cardamom germplasm, which includes cultivars, improved varieties, wild and related
species. Cardamom research, a major thrust area, is hampered by low yield of the
prevailing lines and lack of variability in the population for resistance to devastating virus
diseases. Thus, the productivity of cardamom is very low in India and this is mainly due to
a number of diseases caused by viruses, bacteria, fungi and nematodes.
Conventional breeding methods such as selection and hybridization are being
utilized to increase the spectrum of variation. Evaluation and study of the genetic
variability led to the isolation of few high yielding lines, one of which was released as
CCS-1 (Coorg Cardamom Selection-1). Screening of segregating and irradiated
populations did not yield any promising results with regard to resistance.
Production of diploid homozygous pure lines is a very important step in hybrid
breeding; this is traditionally achieved by many generations of backcrossing to reach
homozygosity. This approach is time consuming and may result in inbreeding depression.
By making use of haploid induction in vitro, with a subsequent doubling of chromosome
number, pure lines can be obtained and incorporated into breeding programmes for
genetic improvement. Thus the anther / microspore culture technology and subsequent
production of dihaploids through microspore callus, is a sure way for production of
hybrids exhibiting maximum heterosis and introduction of variations into the crop.
Furthermore, cardamom is a naturally cross-pollinated crop and the dihybrids from such
hybrids will be recombinant homozygous products useful in the fixation of gene loci.
The work in this project has been undertaken taking these factors into
consideration. The main objectives of this project was production of dihaploids as an
important means by which homozygous lines could be achieved for the subsequent
production of high yielding hybrids exhibiting maximum heterosis. Production of
dihaploids through microspore callus to realize the amount of variations that can be
generated through androgenic callus regenerated haploids and dihaploids and utilize them
for development of disease resistant varieties was also envisaged.
Review of Literature
Haploids are autonomous, sporophytic plants that have gametophytic chromosome
number because they originate from a gametic cell. Haploids are valuable genetic material
in genetic analysis and plant breeding. Natural haploid production has been described in
many angiosperm species, but it is a rare phenomenon. Many attempts have been made to
increase the efficiency of haploid production.
Anther /microspore culture has been widely worked upon, since the initial report
of proliferation of pollen grains in Gingko by Tulecke (1953). This was followed by
reports on direct embryo development from microspores of Datura (Guha and
Maheshwari, 1964, 1966) and development of complete haploid plants in Nicotiana
(Bourgin and Nitsch, 1967). Techniques for culture of isolated microspores was developed
by Nitsch (1974). The history and technique anther culture is reviewed by Maheswari
(1996). Several mechanisms such as parthenogenesis and apogamy, chromosome
elimination and somatic reduction, In vitro culture etc are known to result in haploid
plants (Khush and Virmani 1996).
Since the initial reports of Guha and Maheswari (1966), in vitro haploid
production through anther and microspore cultures have been reported in many species of
angiosperms. Haploids can originate from an egg cell or from a male gamete. It can also
originate from the microspore nucleus before first pollen grain mitosis when pollen or
anther is cultured in vitro. Anther culture is the culture of anthers in nutrient rich media,
under in vitro conditions, to regenerate haploids from pollen grains.
Several reviews on the applications of haploidy in crop improvement have been
published. Among the various methods available for haploid production, anther culture
and chromosome elimination through wild hybridization are the most practical and widely
used. The haploid breeding approach saves time for breeding cultivars through immediate
fixation of genotypes. Although a number of cultivars developed thorugh haploid breeding
have been released for commercial cultivation in rice, wheat, barley and tobacco. Most
promising role of doubled haploids appears to be genome mapping for which they provide
excellent materials to obtain reliable information on location of major genes and QTLs for
economically important traits. The information so obtained should help in increasing the
efficiency of crop breeding programmes (Khush and Virmani, 1996) .
Important factors influencing the performance of anther and microspore culture
have been identified as 1) the genotype of the donor parent, 2) the donor plant growth
conditions, 3) the stage and physiology of microspore development, 4) pre-treatment and
5) culture medium and culture conditions. Mercy and Zapata (1987) studied the effect of
anther orientation and found that most of the anthers plated against the medium callused
on both lobes, however, anthers plated on edge produced callus on the upper lobe.
Toriyama and Hinata (1985) obtained anther calli after one month of culturing panicle
segments with florets at the uninucleate microspore stage whereas, by cutting the tip of
each floret, callus induction occurred within 3 weeks. Sohn et al (1987) also succeeded in
obtaining callus from panicle culture.
In rice, there are two pathways in androgenesis, which lead to pollen
embryogenesis (Sun, 1981; Yang and Zhou, 1979). In the first, the 1st pollen division is
unequal and forms two nuclei � vegetative and generative (both differing in size and
stainability), the vegetative nuclei further divides and forms callus / embryos while the
generative nuclei degenerates. In the second pathway, the first pollen division is equal
leading to two similar nuclei (Chen, 1978)
Cardamom (Elettaria cardamomum Maton)
The cardamom, the spice of commerce, are the dried fruits of a perennial herb
Elettaria cardamomom Maton. The plant is endogenous to South India and Sri Lanka. The
plant has been described in great detail in a monograph by Ravindran and Madhusoodanan
(2001). Cardamom is a tall herbaceous perennial with branched subterranean rhizomes
from which arise several erect leafy shoots and erect or decumbent panicles. There is a
stout horizontal rhizome with numerous fibrous roots in the surface layer. Leafy shoots
are composed of leaf sheaths and are borne in thick clumps. The shoots are 10−20 in
number and 2.0−5.5 m tall. The leaves are distichous with lanceolate acuminate lamina,
25−90 cm wide, dark green and glabrous above, and paler beneath. The panicles emerge
from the rootstock at the base of the leafy shoots and are 60−120 cm long. They are
slender, erect and recumbent or decumbent. The flowers are hermaphrodite, zygomorphic
and about 4 cm long and 1.7 cm across. The bracteole, as is the calyx, is tubular which is
green; shortly three toothed and is persistent. The corolla tube is about the same length as
of the calyx with three narrow strap shaped, spreading, pale green lobes about 1 cm long.
The labellum is composed of three modified stamens, about 1.8 cm long with undulating
edge. There are two rudimentary staminodes and one functional stamen. The stamen has a
short broad filament, with a longer anther, and a connective with a short crest at the apex.
The ovary is inferior, consisting of three united carpels with numerous ovules in axile
placentation and a slender style with a small capitate stigma. The fruit is a trilocular
capsule, fusiform globose, pale green to yellow in colour, varying in size according to the
variety. The fruit contains 15−20 seeds and are dark brown in colour, angled, aromatic,
about 3 mm long and with a thin mucillagenous aril. They contain some white perisperm
and a small embryo. The seed contains some white perisperm and a small embryo
(Purseglove, et al. 1981).
Cardamom is grown at altitudes between 760 m and 1400 m in areas with an
annual rainfall of 1500 to 7000 mm and a temperature range of 10−350C. Cardamom is
very susceptible to wind and require good drainage and cannot tolerate water logging
(Sastri, 1952). The crop thrives best under moderate natural shade (Purseglove et al.
1981).
In India cultivation of cardamom is in the evergreen forests of Western Ghats in
South India, mainly in those regions, which form the natural habitat of the species
(Purseglove et al, 1981), except for a small area in Uttar Kannada and adjoining southern
districts in Karnataka and Wynad District in Kerala where it is grown as a subsidiary crop
in arecanut gardens. In India, cardamom is cultivated in an area of about 81,000 ha, in the
states Kerala, Karnataka and Tamil Nadu with a production of about 10400 tonnes during
1999−2000. India earned a foreign exchange of Rs. 5654.7 lakhs, by exporting 1,100
tonnes of cardamom during 2000−2001 (Source: Director General of Commercial
Intelligence and Statistics-DGCI&S). Currently, Guatemala produces 13,000-14,000
tonnes of cardamom annually. The world production of cardamom is about 24,953 tonnes.
India has a large domestic market for cardamom, consuming about 7000 tonnes per year
(George and John, 1998; Ravindran, 2001).
Cardamom is propagated mostly through seeds and also by vegetative means. The
first crop, which is usually obtained in the third year after planting, is small. Higher yields
are obtained in subsequent years up to the 10th or 15th year (Sastri, 1952).
Gregory (1936) described the basic chromosome number of Elettaria as x= 12 and
the somatic chromosome number of E. cardamomum as 2n = 48. Darlington and Wylie
(1955) also gave the same chromosome number, quoting Gregory (1936). Reports of
Ramachandran (1969) and Sudharshan (1987, 1989) also confirmed the findings of the
earlier workers. However, Chandrasekhar and Kumar (1986) observed variation in
number as well as in the morphology of chromosomes of var. Mysore and var. Malabar
and concluded that aneuploidy as well as structural alterations in chromosomes had
contributed to the varietal differentiation. Meiosis is quite normal and pollen fertility is
high (Sudharshan, 1989).
Palynological studies in cardamom are very much limited. Panchaksharappa
(1966) conducted some studies, and he pointed out that the pollen grains are two celled at
the time of dehiscence. Pollen fertility is reported to be maximum at full bloom stage and
low at the beginning and end of the flowering periods (Venugopal and Parameswar,
1974). Krishnamoorthi et al. (1989) reported that the pollen grains loose their viability
quickly and only 6.5% remained viable upto 2 h and none after 6 h of storage. In vitro
pollen germination studies were reported by Kuruvila and Madhusoodanan (1988). Pollen
germinates in 15% sucrose solution and addition of 150 ppm boric acid improves
germination and tube growth and that the ideal temperature is 15−20oC.
Reports are available on various aspects of cardamom tissue culture. In vitro
methods for clonal propagation of cardamom from vegetative buds have been
standardized (Nadgauda et al., 1983; Priyadarsan and Zachariah, 1986; Vatsy et al., 1987;
Reghunath and Gopalakrishnan, 1991). Kumar et al. (1985) reported the successful
conversion of immature floral buds to vegetative buds and subsequently to plantlets. Many
commercial laboratories are using micropropagation techniques for large-scale production
of cardamom planting material. Field evaluation of tissue cultured plants of cardamom
showed that the micropropagated plants performed on par with suckers (Lukose, 1993).
Sudharshan et al (1997) and Chandrappa et al (1997) have also reported the performance
of tissue-cultured plants. Rao et al (1982) reported plant regeneration from callus cultures
of cardamom. Reghunath and Priyadarsan (1992) reported occurrence of somaclonal
variation in cardamom derived from axenic cultures of juvenile shoot primordia during
their large-scale production.
Rao et al. (1982) reported the successful regeneration of plantlets from callus of
seedling explants of cardamom. Priyadarshan and Zachariah (1986) reported plantlet
formation via adventitious shoots from callus cultures. Protocols for organogenesis and
plant regeneration from rhizome and vegetative bud-derived callus cultures were also
standardized at IISR. This excellent regeneration system (with about 20−50 plantlets per
culture) is being used at present for large-scale production of somaclones and selection of
useful genotypes from them. High variability could be noticed among the somaclones for
the morphological characters in the culture vessels itself. The somaclones are being
evaluated in the field at IISR for realistic estimation of the genetic variability and few
katte tolerant lines could be isolated and they are under advanced stages of screening
(Peter et al, 2001). Cryopreservation of cardamom seeds in liquid nitrogen (LN2) was
reported by Choudhary and Chandel (1995).
No study has been made in India on the anther culture of cardamom, so far. Many
studies have been carried out in the anther culture of cereals and solanaceous plants,
beginning with with classic work of Guha and Maheshwari in 1964. Notable results have
been obtained in anther culture of rice, sorghum, wheat and maize and a few promising
lines from TNAU are being released for large scale cultivation. Two promising lines
developed at CRRI are being evaluated and work on production of high yielding DH lines
inhybrid rice have been intensified.
Materials and Methods
Genotypes Used
Different genotypes of cardamom (Fig.1) have been used in this study to find out
the comparative performance of these genotypes in anther culture. Flower buds from
important genotypes such as CCS1, NKE lines (NKE 3, 9, 27, 34), RR1, MB3, Green gold
were used. Flower buds were collected from plants growing in ideal conditions.
Selection of flower buds
Squash preparations of anthers from flower buds of single raceme were made.
Flower bud size, anther size and stage of microspore development were recorded.
Pollen viability assessment
Pollen was collected from mature flowers prior to opening. Pollen viability was
assessed by staining in acetocarmine, fluorescein diacetate and also by in vitro
germination. Pollen from 10 flowers were pooled and used for viability assessment. In
vitro germination studies were carried out in Brewbaker and Kwack�s medium with
sucrose at 10% concentration.
Explant Collection
Racemes with 3-5 flower buds, at different maturity with anther at different stages
of microsporogenesis, were collected from Cardamom Research Centre (C.R.C),
Appangala, Coorg, Karnataka; Indian Institute of Spices Research (I.I.S.R) experimental
farm, Peruvannamuzhi, Kerala and Sugandhagiri Cardamom Project, Vythiri, Wyanad.
Flower bud from different varieties like Malabar, Mysore and Vazhukka and important
lines from C.R.C, Appangala like CCS-1, NKE-34, RR-1, MB-3 and green gold were
used in the present study. Flower buds were excised and collected in polythene covers.
These covers were tied and kept in icebox and brought to the lab.
Disinfection of Flower Buds
Flower buds collected in polythene covers were taken out and were brushed well
with detergent. The bracts covering each bud were removed with the help of a needle and
washed in running tap water before being surface sterilized with 0.1% mercuric chloride
for 5 min. followed by three washings in sterile distilled water.
After surface sterilization, flower buds were cut of from the raceme with the help
of a sterile surgical blade and forceps and the anther in each bud was dissected out under
sterile conditions. Excised anther was inoculated in different basal media with or without
growth regulators and incubated in dark at 24±20C or under cold as well as hot conditions.
Effect of cold pre-treatment of flower buds
Ccardamom flower buds were given with a cold pre-treatment at 0oC�10oC, prior
to inoculation by keeping in a refrigerator. The Anthers were excised from the cold treated
flower and inoculated on to the medium.
Effect of growth regulators
Different growth regulators like α- naphthalene aceticacid (NAA), 2,4-
dichlorophenoxy aceticacid (2,4-D), indole-3-acetic acid (IAA), indole�3-butyricacid
(IBA), 6-benzylaminopurine (BAP), Kinetin (KIN) and Thidiazuron (TDZ) at different
concentrations ranging from 0.1�10.0 mgl-1 (in the case of TDZ concentrations ranging
from 0.01-0.9 mgl-1 were used) were tested for their efficacy to induce androgenesis. Both
liquid as well as solid media were tested.
The effect of different growth regulators in combinations of different
concentrations was tried in MS medium to study their effect on anther culture (Table ).
Effect of different basal media
In order to study the effect of different basal media, other than MS on cardamom
androgenesis, various basai media like Keller�s, Nitsch and Nitsch (NN) and Schenk and
Hildbrandt (SH) were tried. The growth regulator combinations, which gave responses
when tried with MS basal medium, was tried with all the above basal media (Table).
Effect of liquid media on anther culture
Anthers were cultured in liquid MS media in growth regulator combinations,
which gave responses in solid media, to study the effect of liquid medium. Anthers were
cultured in liquid media in conical flasks and kept on a shaker and also cultured on liquid
media in culture tubes as float cultures and observations were made.
Effect of different additives
To study the effect of different additives on anther culture, different additives such
as as Coconut water (CW) (15-20%), Casein hydrolysate (CH) (0.1-0.2%) and Trypton
(0.1-0.1%) were incorporated along with growth regulators in MS media.
Effect of different carbon sources
In order to study the effect of alternative carbon sources other than sucrose on
androgenesis, carbon sources such as Glucose, Maltose and Fructose were incorporated in
to the MS basal medium and observations were made.
Cold treatment on anther culture
Treatment of cardamom flower buds with a cold shock prior to inoculation was not
suitable as the anthers decayed and turned brown due to bacterial infection if kept for
more than 24 hours after collecting from the plant. Hence the chilling treatment was given
after inoculation on to the medium. The inoculated anthers were incubated in a BOD
incubator at temperatures ranging from 4-160C for 8-72 h. The cultures were transferred to
normal culture conditions after this treatment and observed for further responses.
Nurse culture
Cardamom anthers were cultured, over the cardamom callus separated from the
anther using a sterilized filter paper, on MS media with 0.5 mgl-1 2,4-D+0.1 mgl-1TDZ,
0.2% Trypton along with 25% sucrose and 5% glucose or 15% sucrose and 15% glucose.
Callus induction and proliferation
Anthers were initially cultured in MS medium containing 0.1mgl-1 TDZ and
incubated in dark. The swollen anthers were subcultured to MS medium containing 0.5
mgl-1 2, 4-D and 0.1mgl-1TDZ and observations were made.
Plant regeneration from anther derived callus
Anther derived callus cultures were subcultured on to MS medium with 0.5 mgl-1
2,4-D, 0.1 mgl-1TDZ, 0.2% Trypton along with 25% sucrose and 5% glucose or 15%
sucrose and 15% glucose for plant regeneration.
Hardening and planting out of anther derived plants
Anther derived plants were carefully taken out of the culture vessel, washed to
remove the agar. These plantlets were planted in a mixture of sand and coir pith (1:1) and
kept under 70�80% humidity for 20�30 days. The hardened plantlets were then
established in the nursery.
Results and Discussion
Selection of flower buds
In cardamom the panicles emerge from the rootstock at the base of the leafy shoots
and are 60−120 cm long (Fig. 1a). They are slender, erect and recumbent or decumbent.
The flowers are hermaphrodite, zygomorphic and about 4 cm long and 1.7 cm across (Fig
1b). The bracteole, as is the calyx, is tubular which is green; shortly three toothed and is
persistent. The corolla tube is about the same length as of the calyx with three narrow
strap shaped, spreading, pale green lobes about 1 cm long. The labellum is composed of
three modified stamens, about 1.8 cm long with undulating edge. There are two
rudimentary staminodes and one functional stamen. The stamen has a short broad
filament, with a longer anther, and a connective with a short crest at the apex. The ovary is
inferior, consisting of three united carpels with numerous ovules in axile placentation and
a slender style with a small capitate stigma.
One of the important parameter in anther culture is the selection of anthers at an
appropriate stage of pollen development. In most of the plant species uninucleate stage of
microspore has been reported to give optimal response. There are different methods for
selecting anthers at correct stage of microspore development. Making squash preparation
of one anther of each bud is one such method used in flower buds with more than one
anther. It is not possible in cardamom as it contains only one anther. Flower bud length is
another method for selecting flower buds. Studies were conducted on the relation of
flower size to the stage of microspore development and anther size and the results are
given in Table 1.
Table 1: Relation of flower size to the stage of microspore development and anther size
Nature of flower Flower size (cm) Anther size (cm) Stage of microspore development
I flower (opened) 2.5 0.7 Late uninucleate
II flower (unopened) 1.1 0.5 Early uninucleate
III flower (unopened) 0.6 0.3 Tetrad
IV flower (unopened) 0.4 0.2 MMC
It was found that 1.1 cm long flower buds (Fig. 1c) just emerging from the leafy
bracts contain microspores at early uninucleate stage. These microspores gave better
response upon culture, in that they produced callus and embryoids with a much higher rate
than late uninucleate or tetrad stage (Table 2). Hence, in the subsequent studies anthers
(Fig. 1d), from flower bud of approximately 1.1 cm was used. Flowers of size 4.0 mm
long contain anthers with microspore mother cells (MMC) (Fig. 2a), whereas in flower
size of 6.0 mm showed anthers at tetrad stage (Fig. 2b). Flowers at 2.5 cm size contain
pollen grain at late uninucleate stage (Fig. 2c). In the present study pollen at early
uninucleate stage (Fig. 2d) from flower bud of approximately 1.1 cm was used.
Table 2. Influence of microspore developmental stage on the formation of embryos and callus in
cardamom anther culture
Anthers producing callus and embryos Microspore
developmental stage
No. of anthers
cultured Number %
Late uninucleate 1200 10 0.83
Early uninucleate 1200 30 2.50
Tetrad 500 0 0
MMC 500 0 0
The optimal stage of pollen development is an essential step to increase the
efficiency of anther culture. Routine identification of the stages of pollen development is
at times difficult due to the gradients within the inflorescences. The above result can thus
be used as an indirect method associated with a morphological indicator.
There are a number of stages at which microspores can be diverted into
embryogenesis and these stages vary with species. Tsay and Chen (1984) observed that
the divergence of microspore development to sporophytic pathway lasted 5-6 days in
cultured anthers and could be extended by cold treatment. Calli derived from microspores
at more advanced stages exhibited a lower capacity for plant regeneration (Chen, 1977).
For nearly all perennial fruit crops, microspores at the uninucleate stage have been the
most responsive for induction of androgenesis (Fie and Xue, 1981; Zhang et al, 1990). In
apple, highest rates of callus induction were obtained at very early uninucleate stage,
whereas the induction of embryos occurred at the mid-uninucleate stage, before vacuole
formation (Hofer and Lespinasse, 1996).
Viability of fresh pollen was determined using FDA (Fig. 2e) and acetocarmine
staining and also with in vitro germination studies (Fig. 2f). Location specific differences
were observed in the pollen viability and the average pollen viability ranged from 14% to
72% in different locations (Table 3).
Table 3. The difference in percentage viability of fresh pollen collected from various locations
Location Percentage viability of pollen
IISR, Calicut (cv. Malabar) 14%
Experimental farm, Peruvannamuzhi (cv. Malabar) 30%
C.R.C.Appangala (cv. Malabar) 72%
Sughandhagiri cadamom project (cv. Mysore) 65%
Pollen from C.R.C. Appangala showed highest percentage of viability (72%),
whereas pollen collected from IISR, Calicut showed the least viability (14%).
Palynological studies in cardamom are very much limited. Pollen fertility is reported to be
maximum at full bloom stage and low at the beginning and end of the flowering periods
(Venugopal and Parameswar, 1974). It was observed that the viability of pollen decreased
with increse in storage time. Krishnamoorthi et al. (1989) reported that the pollen grains
loose their viability quickly and only 6.5% remained viable upto 2h and none after 6 h of
storage. The present study agrees with the earlier finding.
Effect of genotype
The different genotypes viz., CCS1, NKE lines (NKE 3, 9, 27, 34), RR1, MB3,
Green gold, belonged to different varieties like Malabar and Mysore were studied for their
response to anther culture. It was observed that the variety Malabar gave maximum
percentage of responding anther cultures (Table 4). In Malabar types all the varieties
responded, whereas in Mysore types out of different genotypes studied only anthers from
variety �Green Gold� responded. The overall efficiency of androgenic callus formation
(1.8%) and embryo formation (0.04%) was very low. In Green gold only callus formation
was observed.
Table 4. Genotypic effect on the induction of androgenic callus and embryos
Geotypes studied % of anthers with callus % of embryogenic callus
Malabar types
CCS 1
NKE lines
RR1
MB3
2.5
1.8
2.2
0.8
0.11
0.05
0.02
0.0
Mysor type
Green gold
0.2
0
Genotype of the donar plant plays an important role in the development of
androgenic haploids (Prakash and Giles, 1992). Genotypic dependence of rice germplasm
has been studied and it was found that certain cultivars and F1 hybrids performed better
than their inbred parents even in ability to form callus which was found to be inherited as
a recessive character conditioned by a single block of genes (Gosal et al, 1996). Genotypic
differences in anther response was reported in apple (Hofer and Lespinasse, 1996),
Brassica species (Duijs et al. 1992) etc.
In the present trials it was found that the variety CCS 1 was most responsive and
the same was used for further anther culture studies.
Effect of growth regulators
The presence of an appropriate concentration of growth regulators in the medium
plays a critical role in callus or embryo formation in anther culture. As there are no reports
on the production of haploids in cardamom, trials were conducted to study the role of
individual growth regulators using MS as basal medium. Both solid and liquid media were
used. The effects are given in Table 5.
Anthers cultured (Fig. 3a) on to media containing different growth regulators
individually did not show any specific response expect in the case of TDZ. The anthers
remained intact for 4-6 days and started browning after that. The anthers were retained in
the same medium for a period of 30-40 days and observed, but did not show any specific
response. When TDZ was used in different concentrations the anthers responded by
getting swollen. Maximum percentage (68%) of response (swollen anthers) was observed
when 0.1mgl-1 TDZ was used in MS medium (Fig. 3b and Table 5). The anthers that
showed a swelling response were retained in the same medium for 30 days and
subcultured on to the same medium and observed for a period of 60-70 days did not show
any other response other than swelling. Studies were conducted to ascertain the effect of
different growth regulators individually when the anthers were cultured on liquid medium,
using the flotation method (Fig. 3c). In liquid medium also the anthers did not show any
specific response to individual growth regulators tried.
Table 5. Effect of individual growth regulators on anther culture of cardamom in MS media Growth regulators
NAA 2,4-D IAA IBA BAP KIN TDZ
Percentage of response
Nature of response
0.5 1.0 2.0 - - - - - - - - - - - - - - - - - - - - -
- - -
0.5 1.0 2.0 -
- -
- - - - - -
0.5 1.0 2.0
- - - - - - - - -
0.5 1.0 2.0
- - - - - - - - - - -
0.5 1.0 2.0
- - - - - - - - - - - - - - -
0.5 1.0 2.0
- - - - - - - - - - - - - - - - - -
0.01 0.05 0.1 0.2 0.5
- - - - - - - - - - - - - - - - - - -
45 68 54 30
Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact Intact
Swollen Swollen Swollen Swollen
The effect of different growth regulators in combinations of different
concentrations was tried in MS medium to study their effect on anther culture (Table 6).
NAA (0.5-1.0mgl-1) when used with 0.5mgl-1BAP alone and along with 0.5-2.0mgl-1KIN
showed only swollen response in the anthers cultured. 2,4-D also produced the same
response when used along with BAP alone or along with BAP and KIN in the same
concentrations. 2,4-D at concentrations 0.5-2.0 mgl-1when used with 0.5-2.0mgl-1KIN
produced friable callus with the maximum percentage of anthers responding in MS
medium fortified with 2.0mgl-1 2, 4-D and 1.0 mgl-1KIN (Fig. 3d). When NAA (0.5-
2.0mgl-1) was used with KIN (0.5-2.0mgl-1) there was production of nodular callus from
the anthers, with maximum percentage of anthers responding in MS medium containing
2.0mgl-1NAA and 1.0mgl-1KIN. When TDZ (0.1mgl-1) was used along with NAA/2,4-D
(0.5-2.0 mgl-1) there was production of nodular callus from anthers with a greater
percentage of anthers responding, the maximum being 54% in MS medium containing 0.5
mgl-1 2, 4-D and 0.1mgl-1TDZ (Fig. 3e).
Table 6. Effect of combinations of growth regulators on anther culture of cardamom in MS media Growth regulators
NAA 2,4-D BAP KIN TDZ
Percentage of response
Nature of response
0.5 1.0 2.0 1.0 1.0 1.0 - - - - - -
0.5 1.0 2.0 - - - - - - - - -
0.5 1.0 2.0 2.0 2.0 2.0
- - - -
- -
0.5 1.0 2.0 1.0 1.0 1.0 - - -
0.5 1.0 2.0 0.5 1.0 2.0 2.0 2.0 2.0 - - - - - -
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 - - - - - - - - - - - - - - - - - -
- - -
0.5 1.0 2.0 - - -
0.5 1.0 2.0 - - - - - -
0.5 0.5 0.5 1.0 1.5 2.0 0.5 0.5 0.5 1.0 1.5 2.0
- - - - - - - - - - - -
0.1 0.1 0.1 0.1 0.1 0.1 - - - - - - - - - - - -
58 60 54 56 51 48 49 41 22 15 - -
36 28 26 54 53 31 21 25 29 32 29 26 14 17 19 27 25 21
Swollen Swollen Swollen Swollen Swollen Swollen Swollen Swollen Swollen Swollen Intact Intact
Nodular callus Nodular callus
Swollen Nodular callus Nodular callus
Swollen Friable callus Friable callus Friable callus Friable callus Friable callus Friable callus
Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus
The majority of the reports available so far suggest that an auxin or a cytokinin has
been required as a component of the anther culture medium (Maheshwari et al, 1982). The
presence of an appropriate concentration of growth regulators in the medium plays a
critical role in callus or embryo formation in anther culture. In some cases, growth
regulators have been required initially for induction but they are not essential for embryo
development. Nitsch (1974) reported that in pollen cultures of Datura presence of growth
regulators was not essential for induction of pollen division whereas Raghavan and
Nagmani (1989) found the best response in the medium containing growth regulators.
Zheng et al (1983) reported an increase in the number of haploid cells and in the
proportion of regenerated plants in the presence of TIBA (2,3,5-tri-iodo benzoic acid).
Sarvesh et al (1993) found that a combination of 2,4-D and Kinetin was more effective.
The studies of Arnison et al (1990) showed that the effect of growth regulators
was cultivar specific and concentration dependant. Besides cytokinins and auxins, the role
fo other growth regulators is not well established. Some studies revealed that gibberellins
have a positive effect (Sapory and Maheshwari, 1976). The effect of absiccic acid on
increased pollen embryogenesis in tobacco anthers (Imamura and Harada, 1980) was
reported and rice anthers (Torrizo and Zapata, 1986). In the present study MS medium
with NAA/2,4-D (0.5-2.0 mgl-1) and supplemented with TDZ (0.1mgl-1) was favourable in
induction of nodular callus.
Effect of different Culture Media
Nutritional requirement of anthers is one of the important factors, which determine
androgenesis.
Table 7. Effect of different growth regulators in three different basal media on cardamom anther culture
Growth regulators Keller�s Nitsch & Nitsch SH
NAA 2,4-D
BAP KIN TDZ % of response
Nature of response
% of response
Nature of response
% of response
Nature of response
0.5 1.0 2.0 1.0 1.0 1.0 0.5 1.0 2.0 - - - - - - - - - - - - - - -
0.5 1.0 2.0 2.0 2.0 2.0
- - - - - - - - -
0.5 1.0 2.0 1.0 1.0 1.0 0.5 1.0 2.0 0.5 1.0 2.0 2.0 2.0 2.0 - - - - - -
0.5 0.5 0.5 0.5 0.5 0.5 - - -
0.5 0.5 0.5 0.5 0.5 0.5 - - - - - - - - - - - - - - -
- - -
0.5 1.0 2.0 - - - - - -
0.5 1.0 2.0 - - -
0.5 0.5 0.5 1.0 1.5 2.0 0.5 0.5 0.5 1.0 1.5 2.0
- - - - - -
0.1 0.1 0.1 - - - - - -
0.1 0.1 0.1 - - - - - - - - - - - -
12 15 10 8 7 5 21 18 16 15 13 13 12 10 9 24 22 19 8 10 11 15 17 18 6 8 8 10 9 7
Swollen Swollen Swollen Swollen Swollen Swollen N.callus N.callus N.callus Swollen Swollen Swollen Swollen Swollen Swollen N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus
21 22 19 12 9 8 31 29 24 25 22 20 23 20 18 28 27 26 14 16 15 19 22 24 9 10 10 15 13 10
Swollen Swollen Swollen Swollen Swollen Swollen N.callus N.callus N.callus Swollen Swollen Swollen Swollen Swollen Swollen N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus
29 32 31 28 25 25 24 22 22 33 32 28 29 26 23 41 38 35 18 21 20 22 24 24 13 16 14 17 14 13
Swollen Swollen Swollen Swollen Swollen Swollen N.callus N.callus N.callus Swollen Swollen Swollen Swollen Swollen Swollen N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus N.callus
The effect different basal media, other than Murashigue and Skoog (MS) were
studied. They include Keller�s, Nitsch and Nitsch (NN), Schenk and Hildbrandt (SH). The
effect of growth regulator combinations, that gave response when tried with MS basal
medium, was tried in these media. The responses were not significantly better than those
obtained in MS medium (Table 7). Comparatively better response was obtained in MS
medium and hence MS medium was selected as basal medium for cardamom anther
culture.
Chu et al (1975) developed N6 medium with reduced ammonium sulphate and
increased potassium nitrate specifically for anther culture. The constituents of basal
medium serve as important factors in eliciting successful androgenesis. The available
literature does not suggest any one culture medium, which could be applicable to all the
systems. The requirements vary from genotype to genotype, however, generally there is an
agreement that the source and amount of total nitrogen as well as kind of growth
regulators are important factors. The basic media generally used, therefore have macro
and microelements, vitamins, sugars and growth regulators (Sapory and Munshi, 1996).
The different basal media commonly used for anther culture are modified from Murashige
& Skoog (MS), Keller�s, Nitsch and Nitsch (NN), Schenk and Hildbrandt (SH).
The usual major salt constituents range from modified Gamborg�s B5 for rice (Cho
and Zapata, 1990), modified Miller�s salts for tobacco (Zarsky et al, 1992) to modified
MS medium for barley (Olsen, 1991). The specific effects of various nitrogen sources
have been studied in barley by Mordhorst and Lorz (1993) who showed the highest plant
regernation with 20-35 mM total nitrogen, a NO3-:NH 4 + ratio of 90:10 and a ratio of
inorganic : organic nitrogen between 90:10 and 71:29. Less attention has been paid to the
effect of the other medium constituents.
Effect of liquid media on anther culture
Anthers were inoculated in liquid MS media in growth regulator combinations,
which gave responses in solid media. Anthers were cultured in liquid media in conical
flasks (Fig. 3c) and kept on a shaker and also cultured on liquid media in culture tubes as
float cultures. Anthers cultures in liquid media did not show any specific response other
than getting swollen.
Effect of different additives
To study the effect of different additives on anther culture, different additives were
incorporated along with growth regulators in MS media.
Table 8. Effect of additives on anther cultures of cardamom in MS medium
Growth regulators Additives
NAA 2,4-D KIN TDZ CW % CH Trypton
Percentage of response
Nature of response
0.5 0.5 1.0 1.0 0.5 0.5 1.0 1.0 0.5 0.5 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - -
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
- - - - - - - - - - - -
0.5 0.5 0.5 0.5 0.5 0.5 - - - - - -
0.5 0.5 0.5 0.5 0.5 0.5
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 - - - - - -
0.1 0.1 0.1 0.1 0.1 0.1 - - - - - -
15 20 15 20 - - - - - - - -
15 20 - - - -
15 20 - - - -
15 20 - - - -
- - - -
0.1 0.2 0.1 0.2 - - - - - -
0.1 0.2 - - - -
0.1 0.2 - - - -
0.1 0.2 - -
- - - - - - - -
0.1 0.2 0.1 0.2 - - - -
0.1 0.2 - - - -
0.1 0.2 - - - -
0.1 0.2
38 39 29 31 37 38 27 30 42 45 34 36 20 21 19 20 28 26 58 60 57 53 63 68 28 31 31 33 39 38
Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus
Additives were used along with the growth regulator combinations that gave
response in terms of nodular callus formation. Additives such as Coconut water (CW) (15-
20%), Casein hydrolysate (CH) (0.1-0.2%) and Trypton (0.1-0.1%) were used (Table 8).
Incorporation of additives did not alter the nature of response of the anthers, but there was
a significant increase in the percentage of anthers responding by producing nodular callus.
Maximum number of anthers responded (68%) in MS medium containing 0.5mgl-1 2,4-D,
0.1mgl-1 TDZ and 0.2% Trypton.
Effect of different carbon sources
Alternative carbon sources other than sucrose such as Glucose, Maltose and
Fructose were also incorporated in to the medium. MS media with 0.5 mgl-1 2,4-D+0.1
mgl-1TDZ+0.2% Trypton that gave best response in terms of nodular callus formation was
used for all experiments with different carbon sources. Alternative carbon sources such as
glucose maltose and fructose were used along with sucrose so as to make the total carbon
source availability as 30%, the above-mentioned carbon sources were also used
individually (Table 9). MS media with 0.5 mgl-1 2,4-D+0.1 mgl-1TDZ, 0.2% Trypton
along with 25% sucrose and 5% glucose or 15% sucrose and 15% glucose produced
shoots along with roots. These shoots developed onto plantlets on transfer to medium
containing 0.5 mgl-1 NAA and 1.0 mgl-1 BA.
Table 9. Effect of different carbon sources on anther culture of cardamom* Carbon source* (gram/l)
Sucrose Glucose Maltose Fructose Percentage of
response Nature of response
30 25 15 5 -
25 15 5 -
25 15 5 -
- 5 15 25 30 - - - - - - - -
- - - - - 5 15 25 30 - - - -
- - - - - - - - - 5 15 25 30
68 65 52 30 11 41 40 30 11 64 58 34 12
Nodular callus N. callus + Shoots N.
callus + Shoots Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus Nodular callus
*All experiments were conducted in MS media with 0.5 mgl-1 2,4-D+0.1 mgl-1TDZ+0.2% Trypton.
Probably the most important of the medium constituents is the carbohydrate source
and concentration and is inevitably linked to the influence of the osmoticum. In barley,
Hoekstra et al (1993) used 0.35 M maltose whereas in wheat the concentration of maltose
was 0.5 M (Tuvusson and Ohlunt, 1993). In another graminaceous member, sucrose at 0.3
� 0.35 M was used for anther culture (Gaillard et al, 1991). The detailed aspects of the
influence of various carbohydrates in barley have been studied by Scott and Lyne (1994).
They concluded that sucrose and glucose had certain toxic effects during the early ohase
of culture.
Effect of cold treatment on anther culture of cardamom
Cold treatment has proved to be beneficial for androgenic induction in many of the
horticultural crops. Treatment of cardamom flower buds with a cold shock prior to
inoculation was not possible as the anthers decayed and turned brown due to bacterial
infection if kept for more than 24 hours after collecting from the plant. To avoid this the
chilling treatment was given after inoculation on to the medium. The inoculated anthers
were incubated in a BOD incubator at temperatures ranging from 4-160C for 8-72 h. The
cultures were transferred to normal culture conditions after this treatment. Cold treatment
did not trigger any specific responses in cultured cardamom anthers.
Culture of Cardamom anthers along with cardamom callus (Nurse culture)
In order to minimize culturing remnants of the anther wall or filament whichmight
lead to diploid / polyploid plantlets, Sharp et al (1972) developed a nurse culture system
for pollen culture. Anthers were cultured along with cardamom callus, separated from the
callus using a sterilized filter paper. Media, in which anthers gave best response when
cultured with out callus, were used in this experiment also (MS media with 0.5 mgl-1 2,4-
D+0.1 mgl-1TDZ, 0.2% Trypton along with 25% sucrose and 5% glucose or 15% sucrose
and 15% glucose). Anthers cultured in this way did not evoke any specific response
except swelling.
Callus formation from anther
From all the above trials a method is devised for anther culture of cardamom.
Anthers were initially cultured in MS medium containing 0.1mgl-1 TDZ and incubated in
dark. Anthers retained their colour for 4�6 days and then turned to light brown within 2�3
weeks of culture. Continued culture in the same medium under the same environment for
about 30 days resulted in swelling of anthers. The swollen anthers on subculture to MS
medium containing 0.5 mgl-1 2, 4-D and 0.1mgl-1TDZ, initiated callus formation and
continuous culture lead to callus proliferation (Fig. 4a).
Pollen derived callus was observed to be of two types. One was nodular callus,
which was compact and white in appearance. Shiny globular bodies were observed on this
type of callus. The frequency of embryogenic callus was less than 1%. The other type of
callus was non-embryogenic, appeared friable and yellowish.
Plant regeneration from anther derived callus
Both embryogenic as well as non-embryogenic callus cultures were subcultured on
to MS medium with 0.5 mgl-1 2,4-D, 0.1 mgl-1TDZ, 0.2% Trypton along with 25%
sucrose and 5% glucose or 15% sucrose and 15% glucose. Continued culture of the
embryogenic calli on this medium resulted in production of shoot and root primordial
(Fig. 4b and 4c). These shoots developed onto plantlets on transfer to medium containing
0.5 mgl-1 NAA and 1.0 mgl-1 BA (Fig. 5a). These plantlets were multiplied on continuous
culture in the same medium. Multiple shoots as well as rooting was achieved in the same
medium (Fig. 5b). The regenerated plants were successfully hardened (Fig. 5c) and
transplanted to the nursery.
In the present study the frequency of anther response and plant regeneration was
observed to be very low and in order to trace the reason for the non response of many
anthers, Squash preparations of the inoculated anthers, at two days of intervals, were made
in FDA (1%) and propionocarmine (2%) to find out the viability and to trace the
development of the microspores. It was found that the viability of the microspore looses
within 4 � 6 days of inoculation and the nuclei of the microspores showed a tendency
towards degeneration after that (Fig. 5d). This may be one of the reason for low response.
Summary and Conclusions
Cardamom (Elettaria cardamomum Maton), known as �Queen of spices�, is a a
perennial rhizomatous plant belonging to the Zingiberaceae family. Its mature and dried
fruits yield the cardamom of commerce. Cardamom is an important spice valued since
time immemorial, for its pleasant flavour and is used directly for domestic and culinary
purposes.
The productivity of cardamom is very low in India due to the low yield of the
prevailing lines and a number of diseases caused by viruses, bacteria, fungi and
nematodes. Conventional breeding methods such as selection and hybridization are being
utilized to increase the spectrum of variation. Evaluation and study of the genetic
variability led to the isolation of few high yielding lines, such as CCS-1, ICRI 1, IISR
Vijetha etc., have been released.
Cardamom, being perennial, development of homozygous lines will take a long
time considering the number of selfing cycles and generations involved. Anther and
microspore culture will provide a short cut for development of haploids and dihaploids
and will reduce the time required considerably. The development of homozygous lines
will further help in better understanding of genetic architecture and inheritance of various
characters in subsequent generations so that better breeding strategies could be developed.
In the present study the primary requisites of anther culture such as optimum age
of panicle and anther suitable for culture, cold treatment procedure, sterilization and
inoculation procedure, photoperiod and light conditions were standardized.
Callus could be induced and proliferated by culturing cardamom anthers initially
in MS medium containing 0.1mgl-1 TDZ and thereafter the swollen anthers on MS
medium containing 0.5 mgl-1 2, 4-D and 0.1mgl-1TDZ.
Pollen derived callus gave plant regeneration in MS medium with 0.5 mgl-1 2,4-D,
0.1 mgl-1TDZ, 0.2% Trypton along with 25% sucrose and 5% glucose or 15% sucrose and
15% glucose. These plantlets were multiplied on continuous culture in the same medium.
Multiple shoots as well as rooting was achieved in the same medium.
These androgenic plantlets have to be cytologically indexed for their ploidy level
to determine their origin and this work is in progress.
However, consistency in development of androgenic plantlets could not be
achieved irrespective of large number of (over 500 ) media combinations tested.
Production of diploid homozygous pure lines is a very important step in hybrid
breeding. By making use of haploid induction in vitro, with a subsequent doubling of
chromosome number, pure lines can be obtained and incorporated into breeding
programmes for genetic improvement. Thus the anther / microspore culture technology
and subsequent production of dihaploids through microspore callus, is a sure way for
production of hybrids exhibiting maximum heterosis and introduction of variations into
the crop.
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ANNEXURE -III
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