Elettaria Cardamomum Maton.) Through Anther Culture / Microspore Culture
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 8800.00 + 1 1,21,440.00 1320.00 (HRA)
Others Nil
Pay of officers Pay TA Other PF Contingency Instt. Total Year of Allow- charges Estab ances lishm ent 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 Opening Amount Amount Balance Balance Sanctioned balance released spent 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
Microspore No. of anthers Anthers producing callus and embryos developmental stage 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 2.5 0.11 NKE lines 1.8 0.05 RR1 2.2 0.02 MB3 0.8 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 Percentage of Nature of response response NAA 2,4-D IAA IBA BAP KIN TDZ 0.5 ------Intact 1.0 ------Intact 2.0 ------Intact - 0.5 ------Intact - 1.0 ------Intact - 2.0 ------Intact - - 0.5 - - - - - Intact - - 1.0 - - - - - Intact - - 2.0 - - - - - Intact - 0.5 - - - - Intact - 1.0 - - - - Intact - 2.0 - - - - Intact - 0.5 - - - Intact - 1.0 - - - Intact - 2.0 - - - Intact - 0.5 - - Intact - 1.0 - - Intact - 2.0 - - Intact - 0.01 - Intact - 0.05 45 Swollen - 0.1 68 Swollen - 0.2 54 Swollen - 0.5 30 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 Percentage of Nature of response response NAA 2,4-D BAP KIN TDZ 0.5 - 0.5 - - 58 Swollen 1.0 - 0.5 - - 60 Swollen 2.0 - 0.5 - - 54 Swollen 1.0 - 0.5 0.5 - 56 Swollen 1.0 - 0.5 1.0 - 51 Swollen 1.0 - 0.5 2.0 - 48 Swollen - 0.5 0.5 - - 49 Swollen - 1.0 0.5 - - 41 Swollen - 2.0 0.5 - - 22 Swollen - 1.0 0.5 0.5 - 15 Swollen - 1.0 0.5 1.0 - - Intact - 1.0 0.5 2.0 - - Intact 0.5 - - - 0.1 36 Nodular callus 1.0 - - - 0.1 28 Nodular callus 2.0 - - - 0.1 26 Swollen - 0.5 - - 0.1 54 Nodular callus - 1.0 - - 0.1 53 Nodular callus - 2.0 - - 0.1 31 Swollen - 0.5 - 0.5 - 21 Friable callus - 1.0 - 0.5 - 25 Friable callus - 2.0 - 0.5 - 29 Friable callus - 2.0 - 1.0 - 32 Friable callus - 2.0 - 1.5 - 29 Friable callus - 2.0 - 2.0 - 26 Friable callus 0.5 - - 0.5 - 14 Nodular callus 1.0 - - 0.5 - 17 Nodular callus 2.0 - - 0.5 - 19 Nodular callus 2.0 - - 1.0 - 27 Nodular callus 2.0 - - 1.5 - 25 Nodular callus 2.0 - - 2.0 - 21 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- BAP KIN TDZ % of Nature % of Nature % of Nature D response of response of response of response response response 0.5 - 0.5 - - 12 Swollen 21 Swollen 29 Swollen 1.0 - 0.5 - - 15 Swollen 22 Swollen 32 Swollen 2.0 - 0.5 - - 10 Swollen 19 Swollen 31 Swollen 1.0 - 0.5 0.5 - 8 Swollen 12 Swollen 28 Swollen 1.0 - 0.5 1.0 - 7 Swollen 9 Swollen 25 Swollen 1.0 - 0.5 2.0 - 5 Swollen 8 Swollen 25 Swollen 0.5 - - - 0.1 21 N.callus 31 N.callus 24 N.callus 1.0 - - - 0.1 18 N.callus 29 N.callus 22 N.callus 2.0 - - - 0.1 16 N.callus 24 N.callus 22 N.callus - 0.5 0.5 - - 15 Swollen 25 Swollen 33 Swollen - 1.0 0.5 - - 13 Swollen 22 Swollen 32 Swollen - 2.0 0.5 - - 13 Swollen 20 Swollen 28 Swollen - 1.0 0.5 0.5 - 12 Swollen 23 Swollen 29 Swollen - 1.0 0.5 1.0 - 10 Swollen 20 Swollen 26 Swollen - 1.0 0.5 2.0 - 9 Swollen 18 Swollen 23 Swollen - 0.5 - - 0.1 24 N.callus 28 N.callus 41 N.callus - 1.0 - - 0.1 22 N.callus 27 N.callus 38 N.callus - 2.0 - - 0.1 19 N.callus 26 N.callus 35 N.callus - 0.5 - 0.5 - 8 N.callus 14 N.callus 18 N.callus - 1.0 - 0.5 - 10 N.callus 16 N.callus 21 N.callus - 2.0 - 0.5 - 11 N.callus 15 N.callus 20 N.callus - 2.0 - 1.0 - 15 N.callus 19 N.callus 22 N.callus - 2.0 - 1.5 - 17 N.callus 22 N.callus 24 N.callus - 2.0 - 2.0 - 18 N.callus 24 N.callus 24 N.callus 0.5 - - 0.5 - 6 N.callus 9 N.callus 13 N.callus 1.0 - - 0.5 - 8 N.callus 10 N.callus 16 N.callus 2.0 - - 0.5 - 8 N.callus 10 N.callus 14 N.callus 2.0 - - 1.0 - 10 N.callus 15 N.callus 17 N.callus 2.0 - - 1.5 - 9 N.callus 13 N.callus 14 N.callus 2.0 - - 2.0 - 7 N.callus 10 N.callus 13 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 Percentage Nature of response of response NAA 2,4-D KIN TDZ CW % CH Trypton 0.5 - - 0.1 15 - - 38 Nodular callus 0.5 - - 0.1 20 - - 39 Nodular callus 1.0 - - 0.1 15 - - 29 Nodular callus 1.0 - - 0.1 20 - - 31 Nodular callus 0.5 - - 0.1 - 0.1 - 37 Nodular callus 0.5 - - 0.1 - 0.2 - 38 Nodular callus 1.0 - - 0.1 - 0.1 - 27 Nodular callus 1.0 - - 0.1 - 0.2 - 30 Nodular callus 0.5 - - 0.1 - - 0.1 42 Nodular callus 0.5 - - 0.1 - - 0.2 45 Nodular callus 1.0 - - 0.1 - - 0.1 34 Nodular callus 1.0 - - 0.1 - - 0.2 36 Nodular callus 1.0 - 0.5 - 15 - - 20 Nodular callus 1.0 - 0.5 - 20 - - 21 Nodular callus 1.0 - 0.5 - - 0.1 - 19 Nodular callus 1.0 - 0.5 - - 0.2 - 20 Nodular callus 1.0 - 0.5 - - - 0.1 28 Nodular callus 1.0 - 0.5 - - - 0.2 26 Nodular callus - 0.5 - 0.1 15 - - 58 Nodular callus - 0.5 - 0.1 20 - - 60 Nodular callus - 0.5 - 0.1 - 0.1 - 57 Nodular callus - 0.5 - 0.1 - 0.2 - 53 Nodular callus - 0.5 - 0.1 - - 0.1 63 Nodular callus - 0.5 - 0.1 - - 0.2 68 Nodular callus - 0.5 0.5 - 15 - - 28 Nodular callus - 0.5 0.5 - 20 - - 31 Nodular callus - 0.5 0.5 - - 0.1 - 31 Nodular callus - 0.5 0.5 - - 0.2 - 33 Nodular callus - 0.5 0.5 - - - 0.1 39 Nodular callus - 0.5 0.5 - - - 0.2 38 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) Percentage of Nature of response Sucrose Glucose Maltose Fructose response 30 - - - 68 Nodular callus 25 5 - - 65 N. callus + Shoots N. 15 15 - - 52 callus + Shoots 5 25 - - 30 Nodular callus - 30 - - 11 Nodular callus 25 - 5 - 41 Nodular callus 15 - 15 - 40 Nodular callus 5 - 25 - 30 Nodular callus - - 30 - 11 Nodular callus 25 - - 5 64 Nodular callus 15 - - 15 58 Nodular callus 5 - - 25 34 Nodular callus - - - 30 12 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. References Arnison, PG, Donaldson, A, Jackson A, Simple C and Keller W A (1990). Genotype-specific
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ANNEXURE -III
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