eChapters

ABSTRACT

Young buds of ginger (Zingiber officinale Rosc.) were isolated and cultured on MS media with BA 3 mg l^-1 induced shoot organogenesis. Young buds from aseptically grown cultures were evaluated for the effectiveness on embryogenic callus production on different concentration of auxin in combination with cytokinin. Among the plant growth hormones tested a combination of 2, 4-D 1 mg l^-1 and BA 0.5 mg l^-1 was most effective in inducing and maintaining embryogenic cultures. The somatic embryo germinated on half strength MS medium supplemented with 3 per cent w/v sucrose and 3 mg l^-1 BA.

Introduction

At the Banana Research Station, Kannara activities focused on Musa germplasm conservation and evaluation are in progress. The aim of the project is to collect and conserve wild and cultivated germplasm representative of the diversity existing in the Musa genepool for evaluation and subsequent use in breeding programmes and to make available selected superior material to the farmers of Kerala. The center presently holds a Musa germplasm of 256 accessions in its field gene bank. These accessions have been characterized and evaluated as per INIBAP/IPGRI Musa descriptors and a database developed.

In a vegetatively propagated crop like banana, in vitro culture is widely employed in germplasm introduction and conservation and is an essential requirement for the safe movement of germplasm (Sharrock and Engels, 1996). By this method, germplasm material is readily propagated, thereby facilitating the dissemination of disease free plants. Since 2001, efforts have been made at Banana Research Station, Kannara to introduce forty new exotic accessions employing in vitro culture for conservation and evaluation. These are material from the global Musa germplasm collection maintained by International Network for the Improvement of Banana and Plantain (INIBAP), France and supplied by National Bureau of Plant Genetic Resources (NBPGR), New Delhi.

ABSTRACT

Tamarind, an economically important multipurpose tropical tree, is a species of choice for sustainable development of wasteland due to its hardy nature and adaptability to various agroclimatic conditions. However, there are very few reports on in vitro regeneration of tamarind. In an attempt to induce caulogenic response, effect of different cytokinins on nodal explants from mature trees was tested. The study extended to seedling explants, revealed that 6-Benzylaminopurine (0.08 - 44.39μM) and Zeatin (0.09 - 9.12μM) induced multiple shoots with callusing at the base of explant. Kinetin (0.09 - 9.29μM) and 6-γ,γ- dimethylallylaminopurine (2iP) (0.09-9.84μM) remained ineffective for multiplication of shoots and callusing was noted at the cut end of explant in contact of medium. Thidiazuron (TDZ) (0.09- 9.08μM) failed to induce morphogenic response and triggered dedifferentiation in the basal cut end. This led to profuse callusing in the explant and restricted morphogenesis. To avoid wound in the explant, tamarind seeds were cultured in medium containing TDZ.

Seedlings were germinated in medium with or without Thidiazuron (4.54, 9.08, 13.12, 18.16μM). Differentiation of the apical meristem to shoot was restricted and proliferation of the cotyledonary node meristem was triggered. Large number of meristematic buds appeared in a radial pattern around the node. The meristematic activity subsequently spread towards the cotyledonary bridge and also in the proximal part of the hypocotyl. These buds differentiated to form shoot primordia and subsequently to shoots in medium devoid of growth regulator. Plants developed by micrografting of these shoots on seedling derived rootstocks, survived in soil.

ABSTRACT

Lack of high yielding varieties, variation among progenies through seedling propagation and transmission of viral diseases through sucker propagation together contribute to the decline in productivity of large cardamom (Amomum subulatum Roxb). To increase the productivity of this crop, planting material raised through tissue culture techniques from few selections having higher productivity was considered. From the study, it was revealed that TC plants were superior to conventionally propagated plants due to vigorous growth during juvenile phase, precocity in the yield and higher productivity compared to that of open pollinated seedlings.

ABSTRACT

In vitro pollination and fertilization could be a tool for heterosis breeding of triploid turmeric cultivars, which are almost non-seed setting naturally. Investigations were undertaken to optimize media combinations for the development of in vitro pollinated ovules to mature seed .The half strength MS medium supplemented with auxins and cytokinins was superior to full strength MS. The supplementation of ½ strength MS medium with either auxins or cytokinins at lower concentrations induced some ovule development. The ovule development was more when auxins and cytokinins together were given. The combination of ½ MS +3% sucrose +NAA 0.5mg l ^-1 +BAP 1mg l^-1 showed maximum ovule swelling in cultures. The combinations of BAP 4mg l^-1 with IAA 0.2mg l^-1 produced callusing instead of ovule swelling.

ABSTRACT

Asuccessful micro grafting technique in Cashew (Anacardium occidentale L.) was developed using in vitro germinated seedling as root stock and shoots from stage II cultures as scions. Grafting success was dependent on the method of grafting, size of the scion and age of the root stock. Among the methods tried, side and cleft grafting were found to be suitable. Due to the ease of operation and faster union, side grafting was preferred. High levels of graft union were obtained when 2 - 3 cm long shoots from stage II cultures were grafted on to 8 - 9 days old in vitro raised root stocks in full strength MS (Murashige and Skoog) medium supplemented with Kinetin (2.5 mg l^-1), NAA (0.5 mg l^-1) and Brassinolide (1mg l^-1). The very high growth of grafted scion was obtained, when the micro grafts were transferred into MS medium containing full strength macroelements supplemented with Kinetin (5mg l^-1), NAA (1mg l^-1) and Brassinolide (1mg l^-1). The micro grafts after a process of hardening could be successfully transplanted into soil. Micro grafting techniques standardized could be used for rejuvenation of shoot explants of mature tree.

ABSTRACT

Cashew (Anacardium occidentale L.) is a woody perennial tree of tropical origin, belonging to the family Anacardiaceae. Cashew is being cultivated in India, Brazil and Africa countries. It is well known for its food, noon food and medicinal uses. In vitro clonal propagation of cashew, which is an effective method for large-scale multiplication of trees was tried. Cashew plantlets were regenerated from shoot explants of 2 - 3 year old grafted plants, which were regularly sprayed with Bavistin 0.1 per cent at weekly intervals. Among the different surface sterilization treatments used, a combination of antioxidants, antibiotics and fungicides was found to be better with around 40 per cent contamination free cultures being obtained. MS medium containing full strength macro elements supplemented with Kinetin (5 mg l ^-1), NAA (1mg l ^-1) and Brassinolide (0.1 mg l ^-1) was found to be best for shoot proliferation and shoot elongation. Micro shoots were rooted in vitro at a frequency of 70-80 per cent, when cultured for 4-8 days in quarter MS liquid medium supplemented with IBA (1mg l ^-1) after the pulse treatment with IBA (100 mg l ^-1) for two minutes. The average number of roots ranged from 3 - 5 with an average length of 1.63 cm. About 50 - 60 per cent of the rooted microshoots survived wearing and produced healthy and vigorously growing plants, which were transferred successfully to the field.

ABSTRACT

Medicinal plants are the most important source of life saving drugs for the majority of the world’s population. Therefore the biotechnological tools are important to select, multiply and conserve the critical genotypes of medicinal plants. With the increase in the demand for the crude drugs, the plants are being overexploited, threatening the survival of many rare species. Also, many medicinal plant species are disappearing at an alarming rate due to rapid agricultural and urban development, uncontrolled deforestation, and indiscriminate collection. Advanced biotechnological methods of culturing plant cells and tissues should provide new means for conserving and rapidly propagating valuable, rare, and endangered medicinal plants. Maintenance of germplasm in the green house or in the field has many difficulties and so there is a need for the in vitro conservation of these plant germplasm. In the present paper we report the in vitro conservation of two medicinal plants and each of it has its own medicinal value. The tuberous roots of Holostemma are medicinally important and are utilized as a major ingredient of the drug ‘jivanthi’. Tylophora is used as a herbal medicine and the plant has been traditionally used as a folk remedy for the treatment of bronchial asthma, bronchitis and dermatitis. Shoot buds of these plants were cultured in Murashige and Skoog’s (1962) medium with different strengths and with different concentrations of sucrose and mannitol. The different treatments induced varied response with respect to the slowing of growth of cultures. Sucrose was found essential in combination with mannitol to induce maximum reduction in the growth of the plant tissues. The cultures could be maintained without sub culturing for over one year and resumed normal growth on transfer to mannitol free medium. This method of conservation has many advantages.

ABSTRACT

Plants as a source of medicine are of special importance in the countries like India which has well developed traditional system of medicine which derive more than 90 per cent of medicaments from higher plants. Several of these also constitute important ingredients of Allopathic medicines. Most of the requirements for the medicinal plants are met from wild sources. But due to the changing ecological factors in the forests and unserupulous collection of medicinal plants, the wealth of medicinal plants is getting depleted and some types have already become extinct. The collection of these plants from the forests cannot cope up with the ever increasing and changing demand from the pharmaceutical, perfumery and cosmetic industry. In order to provide regular and sustained supply of medicinal plants, it is essential now to domesticate and systematically cultivate these plants for which large scale availability of planting material has to be ensured. In this context, in vitro multiplication protocols were standardised for rare, valuable and endangered medicinal plants such as Holostemma ada-kodien, Trichopus zylanicus, Aristolichia indica and Terminalia chebula. The study indicates the feasiblity of rapid multiplication of these plant species using in vitro techniques and the standardised protocols could be used to generate large number of true to type protocols within a short time.

ABSTRACT

In cocoa (Theobroma cacao L.) there are successful reports on the production of shoots from nodal segments under in vitro,the rooting was always inconsistent and mortality during hardening stage was very high. In the present study 20-30 per cent rooting was obtained in half strength MS supplemented with activated charcoal. Maximum rooting was obtained when the shoots were pretreated with IBA 5000 mg l^-1 for three seconds followed by culturing in the media.

ABSTRACT

Aprotocol for in vitro clonal multiplication of Dendrobium var. Betty Ho is reported. Aseptic shoot tip explants cultured in MS medium containing Benzyl Adenine (5 mg l^-1) and casein hydrolysate (1000 mg l^-1) recorded a maximum multiplication rate of 10 fold in 30 days. Explant cultured in LS) medium supplemented with 1-napthalene acetic acid (0.1 mg l^-1) and Kinetinetin (1 mg l^-1) developed a maximum of 8.3 shoots in 30 days. Shoots thus obtained were rooted on MS medium supplemented with BA (0. 1mg l^-1 ) and NAA (0.5mg l^-1). Rooted plantlets after hardening were transferred to green house.

ABSTRACT

Protoplast isolation in orchids is considered to be a valuable tool for introduction of desirable traits through somatic cell fusion. The present work on the protoplast in Dendrobium var.sonia is undertaken with a view to standardize protocol for isolation and regeneration. Tender leaf mesophyll tissues of one-month-old Dendrobium var.sonia were used as explant for protoplast isolation. The leaf tissues were treated with enzyme mixture of cellulase 1per cent (Onozuka -R from Trichoderma viride) and macerase 0.25per cent (from Aspergillus niger) for 16 hours. Protoplast from the Dendrobium var. sonia was successfully isolated and regeneration of shoots and roots were obtained in MS medium containing BA 5mg l^-1+NAA 1mg l^-1.The plantlets were hardened in a span of five months.

ABSTRACT

Cultivated turmeric consists of fertile short duration types with a chromosome number of 2n=84. In vivo crossing was done between two short duration types VK 70 and VK76 to develop a high yielding variety with high curcumin and curing percentage. Seed set and seed development was obtained by in vivo stigmatic pollination. Hybrid seed germination was obtained under in vitro in dark on moist filter paper and the seedlings were micropropagated to establish a population of six plantlets /seedling with in a period of 10 months. The multiple shoots were obtained in the medium of half strength MS + 3 per cent sucrose+ BA 2.5 mg l^-1+ NAA 0.5 mgl^-1. Coupling conventional breeding with tissue culture reduces the breeding time.

ABSTRACT

Cassava, sweet potato and yams are root and tuber crops propagated through vegetative parts. The CTCRI is maintaining a large collection of (3985 accessions) these crop germplasm in field gene banks. Studies were conducted to transfer these collections to in vitro storage so as to clean off systemic infection and protect them from loss due to biotic and abiotic stress. Suitable media protocols, method of surface sterilization, inoculation and incubation were developed for these different crop species in order to establish initial cultures, for their multiplication and storage under slow growth. Meristem, shoot buds and tuber sprouts were used as propagules for culture initiation.Murashige and Skoog (1962) medium was found suitable for all the species studied with variations required in the concentration of growth regulators (NAA, BA and GA ) for meristem culture and micropropagation. Slow growth of cultures was induced using osmotic retardants like mannitol and sorbitol. Addenda like silver nitrate and activated charcoal was found effective in reducing leaf chlorosis and preventing leaf shedding, thereby contributing to culture life and regeneration capacity. The slow growth media developed were effective in prolonging the subculture period to 18 months (cassava), 10 months (sweet potato), and 20 months (yams). The studies have helped in establishing 400 accessions of cassava, 272 accessions of sweet potato and 166 accessions of yams in In vitro Active Gene Bank. The results are presented and discussed.

ABSTRACT

Tinospora cordifolia, a versatile medicinal species with antipyretic and immunomodulatory properties is one of the indigenous sources of the therapeutically valuable alkaloid, berberine. However, the plant contains only meagre amounts of this alkaloid. Hence upgradation of the content of berberine in Tinospora, through biotechnological interventions assumes importance. Four week old leaf, petiole and stem derived calli of two ecotypes of Tinospora viz., Vellanikkara ecotype and Madurai ecotype, subcultured on to Murashige and Skoog (MS) medium, supplemented with various growth regulators, were incubated in light and screened for the presence of berberine employing thin layer chromatography. MS medium with NAA @ 2 mg l^-1 supplemented with BA or kinetin, each at 2 mg l^-1, was identified as the basal production medium for in vitro production of berberine, yielding 7.55 μg and 7.36 μg berberine respectively, per gram of calli. Calli produced from stem segments registered maximum amount of berberine compared to leaf and petiole derived callus cultures. Increasing levels of sucrose in basal medium did not have a favourable influence on the expression of berberine in the test calli. Reducing nitrate and phosphate content of the basal medium to half their strength resulted in enhanced berberine production in in vitro cultures of both the experimental ecotypes. Favourable influence of precursor feeding on upgrading the content of berberine in leaf and stem derived calli of both the ecotypes of the experimental species was also confirmed in the study.

ABSTRACT

Ophiorrhiza mungos is a rich source of the potent antineoplastic therapeutic agent Camptothecin, which is effective in the complete remission of lung, breast, uterine and cervical cancer. With increasing demand, most of the plants have been indiscriminately exploited from their natural habitat for the isolation of this valuable therapeutic agent. The biotechnological application such as plant tissue culture seems to be a viable option for the production of this high value therapeutic compound without destroying the natural flora. For this purpose various tissue culture techniques have been established to enhance the production of this compound from different parts of O. mungos. The present study revealed that the Physical elicitation with varying doses of irradiation is significantly higher (0.87mg/ g dry weight) than that obtained by chemical elicitation using methyle jasmonate (0.41mg/ g dry weight) compared to control. Biotic elicitors like chitin, chitosan, fungal and bacterial extracts were also used and considerable increase in the production of Camptothecin was obtained in all these cases. Permeabilization with Tween 20 found to be enhancing the exudation of camptothecin (0.021 mg/ml) compared to control (0.0003mg/ml). Hence, the present study gives an alternative for large scale production of camptothecin without affecting the natural flora in a cost effective method.

ABSTRACT

Mucuna pruriens (L.) DC. is one of the important medicinal plants that contains L DOPA (L-3,4 dihydroxy phenylalanine) which is the precussor for the neurotransmitter dopamine that plays an important role in curing Parkinson?s disease. To accommodate the huge demand for L DOPA, in vitro production of the drug using cell cultures is felt essential. Hence, an experiment was carried out with completely randomized design with seven treatments viz., T₀ - Control; T₁ - MS + 2 mg l^-1 NAA; T₂ - MS + 2 mg l^-1 IAA; T₃ - MS + 2 mg l^-1 2,4-D; T₄ - MS + 2 mg l^-1 Kinetin; T₅ - MS + 2 mg l^-1 BA; T₆ - MS + 1 mg l^-1 IAA + 1 mg l^-1 BA and 3 replications. Among the various explants investigated stem bits registered the least days (12.38) for callus induction. The stem bits responded well with better callusability (78.57 per cent), relative growth rate (2.57) and callus index (249.16). MS medium + 2 mg l^-1 2,4-D was found to be the best for inducing callus in various explants namely stem bits, leaf bits and root bits. L DOPA accumulation was more in cell suspension cultured in liquid MS medium with 4 per cent sucrose + 1 mg l^-1 IAA + 1 mg l^-1 BA and that was confirmed by means of HPLC.

ABSTRACT

Protoplast isolation of Piper nigrum L.and Piper colubrinum Link. was attempted using axenic leaves at two different osmotic potentials viz. 0.60 and 0.65 molar (M) osmoticum. In Piper nigrum, highest protoplast yield (6.90 x 10⁴ protoplasts ml^-1) was observed in the enzyme combination 1.40 per cent cellulase and 0.310 per cent pectinase while in Piper colubrinum, highest protoplast yield (13.0 x 10⁴ protoplasts ml^-1) was observed in the enzyme combination 1.0 per cent cellulase and 0.217 per cent pectinase during 21 h of digestion. In both the species, 0.6 M osmoticum was found to be optimum to maintain the osmotic potential of the isolation solution. Filtration-centrifugation technique was found to be superior in purifying the protoplasts of Piper species compared to sucrose floatation method. Centrifugation at 1000 rpm for three minutes was found to be best in purifying Piper nigrum protoplasts. For purifying Piper colubrinum protoplasts, 600 rpm for three minutes was found optimum. The protocol developed for protoplast isolation in Piper species can be used for biotechnological means like somatic hybridization between the two and gene transfer to develop foot rot resistant black pepper lines.

ABSTRACT

Haploids are of great importance in breeding programmes for the production of homozygous diploid lines. This technique reduces the time span in developing new varieties and fixes the traits including recessive ones in the homozygous condition. The haploid embryos or doubled haploid plants could be used in mutation breeding, genetic engineering, biochemical and physiological studies. Isolated microspore culture for the production of haploids has not yet been reported in Hevea brasiliensis. Hence the present work was initiated with the view of producing haploid plants by the culture of isolated pollen grains. The flower bud of the clone RRII 105 at the late uninucleate to early binucleate stage was used for culture initiation. Different pretreatment solutions were tried at different temperatures (4°C, 27°C and 33°C) with the macronutrients of N6 media. Three kinds of callus induction media were used viz. N6, WPM and Hunters medium supplemented with myoinositol (200 mg^-1), glutamate (150 mg^-1), phenyl acetic acid (7.0 mg^-1), sucrose (70 g^-1) and growth regulators. Induction medium include moving liquid, static liquid and semi- solid medium. The ovary co-culture was also tried in the induction medium to increase the frequency of callus formation. Pretreatment of the anther with 0.3 M mannitol at 33°C promoted the division of microspores. Static liquid cultures improved the formation of multicellular microspores compared to semi-solid or moving liquid cultures. N₆ basal medium supplemented with 2.0 mg/l 2.4-D and 1.0 mg/l KIN induced microcalli formation. When the ovary co-culture was included in the callus induction medium, the rate of cell proliferation was increased considerably and the duration of callus formation was reduced from 3 months to 14 days. The results of the present study throws light towards the possibility of regenerating doubled haploids in future.

ABSTRACT

Rose (Rosa hybrida Linn.) is universally acclaimed as the ‘Queen of Flowers’. But this woody plant is considered less amenable to in vitro culture as compared to herbaceous plants. This is due to the fact that the establishment rose explants is difficult due to heavy phenolic exudation. In the present study, this problem was solved by a very simple and cost effective method. The cut ends of the explant were sealed with wax after thorough sterilization. Ten different explant collection periods with 20 different hormonal combinations in MS culture medium were taken for establishment. The winter dormancy period (Mid- December) was considered best for explant collection and showed 85% survival. Amongst the hormonal combinations 5mg/l BA coupled with 0.05mg/l NAA was considered the best. The average number of leaves (7.5) and average shoot length (7.3 cm) was also highest in this treatment, 15 days after culture establishment.

ABSTRACT

Attempts were made to induce variability by irradiation of axillary buds of rose cv. Folklore followed by in vitro culture. Axillary bud explants collected at different growth stages were subjected to gamma irradiation at different doses (20, 30 and 40 Gray). The in vitro induced micro shoots were sub cultured on a rooting medium (M.S. basal medium supplemented with IAA 1.00 mg/l + NAA 1.00 mg/l + activated charcoal 500 mg/l). The rooting percentage was the lowest in explants irradiated with 40 Gy. Rooting efficiency was severely affected when buds were collected at vegetative phase. Significant interaction between bud stages and gamma rays was noticed for days to root initiation and number of roots per culture. Gamma irradiation at 30 and 40 Gy significantly delayed root initiation at all stages of buds exposed. Significant reduction in number of roots/ culture, and root length was observed at higher doses of gamma exposure. The reduced rooting efficiency may be due to inhibition of cell division on account of chromosome damage.

ABSTRACT

Wood apple is a hardy tree grown in arid and semiarid regions of the country. It is generally propagated through seeds. The traditional methods like budding and inarch grafting require 9 to 12 months for the root stock to come buddable size and thus 1½ to 2 years to become ready for transplanting. The present study was initiated to prepare a grafts within eight months by adopting the micrografting technique. The seeds were sown in the month of April in polybags filled with FYM, sand and soil in equal proportions with a pinch of blitox. The micrografting was done on 1½, 2, 2½ and 3 month old seedling root stock having a diameter of 0.13 to 0.15 cm, 0.17 to 0.19 cm, 0.20 to 0.22 cm and 0.23 to 0.26 cm, respectively. The seedling root stocks were decapitated with the surgical blade and 1 to 2 cm deep split was made. The scion micro bud (1 to 2 cm length and 1 to 2 mm width) of previous season’s growth was inserted in the split. The micropipette cap (0-200 μl) was inserted on the grafted portion. After sprouting of the micro bud, the micro pipette cap was removed. The study revealed that, micro bud grafting success was significantly influenced by the root stock age. Early bud take was observed when the micrografting was done on 2½ month (10.73 days) and 3-month (11.00 days) old root stock. The percent bud take on 3-month old root stock was significantly higher (99.33 per cent) than the rest of root stocks. Micrografting done on 3-month old seedling root stocks in June-July is suggested for quick multiplication of woodapple.

ABSTRACT

To study the influence of season on explant collection, axillary buds from mature trees of three elite genotypes of tamarind viz., Sour pulped type, Red pulped type, and Sweet pulped type (Thailand) were collected monthwise from September, 2002 to August, 2003, sterilized properly and cultured in MS medium supplemented with BAP (1, 1.5, 2, 2.5 and 3 mgl^-1) + GA 0.5 mgl^-1 + NAA 0.1 mgl^-1. The studies revealed that, the bud break percentage was highly dependent on the season of explant collection. In case of Sour pulped type, bud break was recorded during the month of April, May and June. Maximum per cent bud break (50.00 per cent) was recorded in May and June but least number of days were required for bud break (12.33) in April month. In Red pulped type, the bud break was observed during February to June. The axillary buds collected and cultured during the month of February recorded the best results with respect to the per cent bud break (100 per cent) and least number of days taken for bud break (7.12 days). In Sweet pulped (Thailand) type, bud break was recorded during the months of February to June. The axillary buds collected and cultured in the month of may recorded the best results with respect to the percent bud break (87.50 per cent) but March showed least number of days for bud break. Nodal segments cultured on MS + BAP 2 mgl^-1 + GA 0.5 mgl^-1 + NAA 0.5 mgl^-1 gave maximum and early bud break in three genotypes, but sprouted shoots showed abscission of leaflets and died later on.

ABSTRACT

Cycas is the largest genus among cycads - the surviving remnants of an ancient line of plants of the early Mesozoic period. The IUCN has classified more than half of the 182 species of Cycadales as endangered, vulnerable or rare. The use of in vitro techniques could permit a way to conserve many of the endangered species. None of the in vitro studies has been successful in establishing a functional protocol for artificial propagation of cycads. The objective of the present investigation is to explore the possibilities of in vitro plant regeneration from megagametophyte explants of Cycas circinalis - the only species of Cycas grown in Kerala. Megagametophyte from the ovules of ten-year-old Cycas circinalis plant was used as explant. Standard tissue culture techniques were adopted for the in vitro culturing of the explant. The basal medium involved major salts of B5 and minor salts of MS media. Glutamine, casein hydrolysate, coconut water and megagametophyte extract were used as supplements. The plant growth regulators - Kn, BAP, 2iP, IAA, NAA and 2,4- D - were incorporated alone and in combinations. Highest percentage of shoot induction and highest average number of shoot buds per culture were observed in PGR combination involving Kn + BAP along with one of the auxins (2,4-D or NAA or IAA). Shoot buds developed did not show further growth even after one year. The protocol employed in the present investigation may lead to multiple shoot proliferation and help in large-scale propagation of Cycas circinalis.

ABSTRACT

In Kerala, the commercial cultivation of tomato is limited due to the bacterial wilt disease caused by the soil borne pathogen Ralstonia solanacearum. The release of bacterial wilt resistant varieties like Sakthi, Mukthi and Anagha has increased the area under tomato. But, these varieties are susceptible to tomato leaf curl virus diseases caused by Germini group virus and transmitted by white fly (Bemisia tabaci). The disease incidence varies from 10.77% to 90.5% and the yield loss is tremendous. Unless the tomato variety resistant to ToLCV disease is resistant to bacterial wilt also, it cannot be recommended for cultivation in the State.

The somaclonal variations have been well exploited in developing disease resistant varieties in many crops. Therefore an experiment was formulated to study the somaclonal variations and to screen the somaclones for resistance to tomato leaf curl virus disease in L. esculentum var. Sakthi and L. pimpinellifolium.

The somaclones of Sakthi and L. pimpinellifolium along with the parental lines were planted in earthen pots containing potting mixture with bacterial wilt sick soil. These were screened for resistance to ToLCV disease by artificial inoculation of the vector Bemisia tabaci inside the cages. The observations were recorded on ex vitro survival, biometric characters and reaction to ToLCV and other major diseases.

The ex vitro survival was 46.2% in Sakthi and 36.6% in L. pimpinellifolium. The somaclones of Sakthi showed wide variation for plant height and spread compared to that of L. pimpinellifolium. Some of the somaclones exhibited ‘gigantism’ with potato leaved plants. The yield of the somaclones of Sakthi were higher than that of parental line.

ABSTRACT

The in vitro culture studies in crop plants show that there is a differential response of various explants to different concentrations of auxins and cytokinins. This indicate that hormonal requirement is specific to the genotype and developmental stage of the explant. Therefore standardisation of media and explants were done for in vitro callus culture and plant regeneration in L. esculentum and L. pimpinellifolium at the Department of Olericulture, College of Horticulture during 2000-2002.

The source materials selected for the study were L. esculentum var. Sakthi and L. pimpinellifolium. The explants were leaf segments (0.5 cm²) from fully opened leaves, nodal segments (0.5 cm) and internodal segments (1.0 cm). The media consisted of the basal MS and ½ MS media along with different auxins and cytokinins either singly or in combination. The auxins used were NAA (0, 1, 2 and 3 mg l^-1 and 2,4-D (0.5, 1, 1.5 and 2 mg l^-1) and the cytokinins used were BA and kinetin (1, 2, 3, 4 and 5 mg l^-1).

ABSTRACT

The tissue culture derived black pepper plants of variety Panniyur 4 were compared with conventional propagules for its vegetative growth and field performance. The in vitro derived plants recorded better rooting, survival rate and shoot growth than the conventional propagules. More than 90% of the TC derived plants recovered after primary hardening and 99% recovered after secondary hardening, while it was only 70% in conventional propagules at both the hardening stages. The secondary roots developed in TC derived plants were observed to be far more superior in number and length. The initial growth rate in terms of plant height, number and size of leaves was much better in TC derived plants. The TC derived plants developed numerous bearing shoots - laterals - in the first year of growth and started bearing in the first year itself. The performance was superior to conventional propagules in which production of laterals and bearing begins much later. Thus the performance of TC derived plants was rated as superior to conventional propagules.

ABSTRACT

The tomato TYLCV tolerant line MP-1 which has high efficiency of crossability & dwarf mutant dg/dg (Manapal) were choosen for the study of enhanced production of plantlets through tissue culture. The cotyledonary explants, hypocotyl sections & decapitated seedlings were taken to assess the regeneration efficiency of the explants as a base for efficient genetic transformation. The decapitated seedlings regenerated much faster (9.3 days) putting forth new sprouts of shootlets which were then transferred to rooting medium. Whereas cotyledonary leaves showed regeneration of plantlets (18.3 days) which is almost double the duration of decapped seedlings. The hypocotyl sections proliferated into callus & regenerated in a much slower rate (27 days). Hence, the faster rate of regeneration from decapitated seedlings served as a efficient base for enhanced production of plantlets during transformation studies. Also the number of harvest of new sprouts (2.3 regenerations) from decapitated seedlings is an added advantage for deriving more plantlets from single seedlings.

ABSTRACT

Multiple shoot induction from the cotyledonary node explants of Leucaena leucocephala was studied in presence of various concentrations of TDZ. With the increase in concentration of TDZ from 0.045 μM to 0.227 μM the number of regenerated shoots increased. However, the length of individual shoot decreased. Further increase in TDZ concentration upto 22.71 μM resulted in the formation shoot clusters. Presence of TDZ in the medium inhibited shoot elongation. Transfer of the regenerated shoots to ½ x MS medium supplemented with 0.54 μM NAA ensured elongation of the shoots, which also developed roots. Rooted plantlets were transferred to coco peat for further development and hardening. This highly efficient regeneration protocol may find application in the genetic transformation of this leguminous tree species.

ABSTRACT

Myriophyllum aquaticum (Velloso) Verdcourt, commonly known as ‘parrot feather milfoil’ is the aquatic plant belonging to the family Haloragaceae. The plant gets the name ‘parrot feather’ from its feather-like leaves arranged around the stem in whorls of 4 to 6 giving it the value of a beautiful aquarium plant. It is a commercially valuable imported aquatic plant used in aquarium plant trade. These plants are exclusively females and as such no seeds are produced. Moreover, current macropropagation techniques are insufficient to produce sufficient propagules for the domestic and export market. Tissue culture techniques offer great opportunities in this regard and attempts were made for micropropagation of this valuable species. A viable protocol has been outlined for the in vitro mass multiplication of Myriophyllum aquaticum. Shoot regeneration was induced from nodal segments and leaves in half strength Murashige and Skoog (1962) medium supplemented with 1.0 mg l^-1 benzyl adenine. Incorporation of 0.2 mg l^-1 kinetin along with benzyl adenine in the culture medium resulted in a marked increase in the frequency (1:20) of multiple shoot formation. The in vitro raised plantlets were successfully rooted, hardened and planted out. The protocol is ready for commercialization.

ABSTRACT

The mass production of orchids through in vitro approach is now practising on a commercial scale. But, in most cases, it is being done via an intermediate callus which may lead to variability in the progenies. A study was made to standardize a protocol for rapid mericloning of tropical orchid dendrobium cv. sonia from the shoot tip explant without an intermediate callus so as to produce true to type progenies. The efficiency of production of clones through agar gelled medium and shake cultures were compared. The shoot tip explants produced an average of 6-8 multiple shoots on agar gelled (0.8% w/v) MS medium containing 3% sucrose as carbon source supplemented with a hormonal combination of 0.75 mg l^-1 NAA and 2.5 mg l^-1 KIN without an intervening callus, within a period of 3 - 4 months. When the shoot tip explants were cultured in agitated ( 80 - 100 rpm) liquid Vacin and Went ( VW) medium supplemented with 3% sucrose, 2.0 - 2.5 mg l^-1 BA and 0.5 - 1.0 mg l^-1 NAA could induce a large number of protocorms within a period of two weeks. Agitated liquid MS medium supplemented with 0.5 mg l ^-1 IBA and 2 mg l^-1 BA or 0.5 mg l ^-1 2,4 - D, 0.5 mg l^-1 GA₂ and 3.0 mg l^-1 BA was identified as the rapid multiplication medium of the protocorms. About 60 - 80 protocorms could be produced from a single protocorm within a period of 3 weeks. Transfer of these protocorms to agar gelled (0.8%w/v) MS medium containing 2% sucrose supplemented with 20 - 25% coconut milk (CM) was found ideal for plantlet formation of size 1.0 - 1.5 cm within a period of three weeks. Sub culturing of the plantlets to the same medium resulted in rapid profuse rooting. Transfering the rooted plants into bottles containing sterile coconut husk provided with half MS medium without sucrose and hormones was sufficient for in vitro hardening. By this two stage culturing technique, about 50 - 60 true to type clones can be produced from a single shoot apex within a period of 3 months.

ABSTRACT

Considering the difficulties faced during ex vitro establishment of orchid plants the present investigation was undertaken to elicit information on the influence of different sucrose levels in the in vitro rooting medium and by subjecting the plants to different levels of triazole at the time of planting out in order to overcome the mortality percentage at the time of ex vitro establishment. Among the growth parameters observed viz. crop growth rate, net assimilation rate, and relative growth rate was found to be high in plants treated with 40 g/l of sucrose concentration and also at the triazole level of 5 mg/l. The photosynthetic rates were observed to increase meanwhile the transpiration rate was declined in plants grown under the above treatment levels of sucrose and triazole. The maximum survival percentage was observed when the in vitro plants grown in rooting medium with 40g/l of sucrose which is attributed to the influence of sucrose concentration on morphological characters studied viz. plant height, number of leaves per shoot and number of roots per shoot. Triazole treatment of plantlets at the time of planting out helped in better survival percentage which is the whole sum effect of increase in number of leaves per shoot, number of roots per shoot of plants grown under the triazole level of 5 mg/l. Where as the height of the plant was inversely proportional to triazole concentration,the biometric aspects studied viz root length, total fresh and dry weight of the plants with the above mentioned treatments were found to be superior. The present study envisages the measures to be taken during in vitro propagation and ex vitro establishment of tissue cultured plants in order to over come the field mortality.

ABSTRACT

Astudy was conducted to establish the regeneration capacity of pseudobulb segments of a Dendrobium hybrid orchid (D. nobile L. x D. chrysotoxum L.) in vitro on full strength (MS) and half strength (½ MS) Murashige and Skoog medium. Pseudobulb segments having 0.3-0.5 cm length cut from the in vitro maintained hybrid orchid were inoculated on both media supplemented with different concentrations and combinations of NAA, BAP and Kn. On MS medium the pseudobulb segments failed to respond, however, in ½ MS medium the explants responded well. Responding explants were maximum (35%) on the medium ½MS + 0.1 mgl^-1 NAA + 0.5 mgl^-1 BAP. An average of two (2) shoot buds per explant were regenerated on ½ MS + 2.0 mgl^-1Kn and ½ MS + 0.1 mgl^-1 NAA + 1.0 mgl^-1 BAP. The regenerated plantlets grew healthily on subculture after separation from the pseudobulb segments.

ABSTRACT

Asimple and reproducible protocol for the rapid propagation of Clitoria ternatea L., a flowering garden climber cum medicinally valuable plant, was developed through somatic embryogenesis from the leaf explants. Leaf segments excised from 12 days old in vitro raised seedlings on inducing callus by culturing in MS medium containing 0.5 mgl^-12,4-D and 0.1 mgl^-1 BAP and sub-culturing callus to MS medium containing 1.0 mgl^-1 BAP, produced numerous somatic embryos within 30 - 45 days. Embryos germinated and produced plantlets with intact shoot and root when they were cultured in MS medium devoid of any hormones and supplemented with GA₂ 0.1 to 1.0 mgl^-1. Elongation of plantlets could be achieved on hormone free MS medium in about 3 - 4 weeks. Well- grown plants on transfer to pots, containing sterile sand, in a growth chamber and three weeks later to the greenhouse gave about 40 per cent survival.

ABSTRACT

Successful in vitro clonal propagation of mature tree of Lasoda (Cordia myxa Roxb) has been achieved through single node explant. Both types of organogenesis of shoot and root formation were obtained by placing nodal explant on shoot induction medium and subsequently on rooting medium. More than 90% culture were responsive for in-vitro axillary shoot proliferation on MS media supplemented with 30g Sucrose 8.0 g agar and a combination of 2.0 mg BA and 0.1 mg NAA per litre. After culture period of 6-8 week, the nodal explants with micro shoot were rooted on the MS media supplemented with30 g Sucrose + 3g activated charcol and different level of NAA (0, 1, 3 and 6 mg/ litre). The season and position of explant on the mother plant was also shown to influence in vitro performance of the regeneration in terms of establishment of aseptic culture, percentage of responsive explants and intensity of leaching of growth inhibitory phenolic chemicals.

ABSTRACT

Aloe vera is one of the oldest known therapeutic herb belongs to the family Liliaceae commonly known as guar patta. Gel from fleshy leaves of Aloe vera is rich in 200 nutrients which are used in healing the burns, wounds, gastric ulcers, treatment of diabetes and also used in normal functioning of the liver. Aloe vera is a desert plant which needs water in very less quantity. As Aloe vera is second in ranking in drug production in the world. Government of India is also trying to promote Aloe vera as a crop in dry and semi dry regions of India. As it needs very less and cheaper inputs, Aloe farming promises higher economic gains to farmers. But as the plant grows vegetatively the quality planting material is a problem. Tissue culture provides an opportunity in production of quality seedlings. To optimize the regeneration system in Aloe vera, MS based media were tried to induce shoot formation in cultured shoot tip explants. 6-10 cm long offshoots were collected. Shoot tip explants were cut vertically after sterilization in laminar hood and cultured on MS basal media supplemented with various growth regulators. A total of 23 media were tried for shoot regeneration. All the cultures were incubated under fluorescent light. Different concentrations of BAP (2, 5, 10 mg/l) were used and MS medium with 2 mg/l or 5 mg/l BAP exhibited similar response while BAP at 10 mg/l showed poor shoot formation. TDZ when used at 1 mg/l concentration improved percent shoot formation marginally. Multiple shoots were observed on TDZ containing medium and BAP (2.0 mg/l) supplemented medium in 12-15 days. Multiple shoots were obtained in 12 days when 2 mg/l BAP was added along with 0.18 mg/l IAA. The number of multiple shoots ranged from 4 -15 per explant. The regenerated plants developed roots on MS medium supplemented with 0.1mg/l NAA.

ABSTRACT

In vitro clonal propagation of Smilax zeylanica Linn was attempted. Axillary buds from shoot tip and single node explants were induced to proliferate on half strength MS basal medium supplemented with BA (0.5, 1.0 and 2.0 mg l^-1) and IAA (1.0 and 2.0 mg l^-1). Multiple shoots (6.0 - 10.0 per explant) developed from the axillary buds in about four weeks time in all the treatments tried. However, the highest rate of multiple shoot production was in the medium containing BA 1.0 mg l^-1 and IAA 1.0 mg l^-1. Maximum shoot length was also observed in the same medium. Rooting (83.3 per cent) of micro shoots could be induced on MS supplemented with NAA 0.5 mg l^-1. Individual rooted plantlets could be successfully transferred to soil and established in the field with 80 per cent survival.

ABSTRACT

Ipomoea mauritiana (L.) belonging to the Family Convolvulaceae is an important medicinal plant. The roots, leaves and flowers possess medicinal properties. It is used in indigenous medicine as a tonic, aphrodisiac, galactogogue, diuretic, carminative, expectorant and appetizer. It is useful in syphilis, inflammation, leprosy, diseases of blood and in the improvement of voice and complexion. Tender nodal portions from one year old disease free plants grown in pots were used as the source of explants. The explants were inoculated on half strength MS medium containing 3.0 per cent sucrose and 0.6 per cent agar supplemented with 2,4-D 0.5 mg l^-1. All the explants showed callus initiation after three weeks. The contamination free, one week old callus cultures were used for testing the effect of the different treatments. The treatments comprised of different combinations of BA (0, 1.0, 2.0, 4.0 and 8.0 mg l^-1) and NAA (0 and 0.1 mg l^-1) added to half strength MS medium. The highest rate of shoot regeneration was obtained on half strength MS medium supplemented with BA 4.0 mg l^-1. Rooting of the proliferated shoots could be induced on the same basal medium supplemented with IBA 1.0 mg l^-1 and NAA 1.0 mg l^-1. The regenerated plants were hardened in a humidity chamber and successfully transferred to pots, with 90 per cent survival.

ABSTRACT

In vitro propagation of Plumbago rosea was attempted. One year old plants were used as the source of explants. Nodal explants were inoculated on half strength MS medium supplemented with 2,4-D 1.0 mg l^-1. Induction of callus was observed three weeks after inoculation. The calli were sub-cultured on half strength MS medium supplemented with BA (0.5, 2.0, 3.0 and 4.0 mg l^-1) and IAA (0.0 and 1.0 mg l^-1). Eight treatments with six replications were tried. Benzyl adenine 2.0 mg l^-1 promoted the proliferation of shoots (23.2). Rooting of micro shoots was obtained on MS medium supplemented with IBA 1.5 mg l^-1. The plantlets were then transferred to pots containing sand/soil mixture (1:1) and kept in a mist chamber. The survival rate was found to be 90 per cent.

ABSTRACT

Centella asiatica (Linn.) previously Hydrocotyle asiatica belongs to the family Apiaceae.It is commonly called gotu kola and is rich in triterpene glycosides such as asiaticoside and madecassoside. Health claims range from enhanced memory and longevity, to the treatment of leprosy. It is also used in the treatment of various skin diseases, ulceration, chronic rheumatism, malaria, fever, epilepsy and enlargement of glands. Inspite of its wide range of uses,it is still collected from the wild and commercial cultivation is lacking. A good tissue culture protocol therefore would be useful for commercial propagation and thereby saving the plant from extinction. Of the various explants, nodal segment gave the best response. For direct shoot regeneration from nodal segments, BAP 2.0 mgl^-1, and BAP 1.0 mgl^-1 with Kinetin 1.0 mgl^-1 was found optimum. Callusing was observed in nodal segments and leaf bits when 3.0 mgl^-1 NAA along with 0.3 mgl^-1 BAP was used. ½ MS medium supplemented with IBA 0.5 mgl^-1 and NAA 0.2 mgl^-1 induced rhizogenesis of the regenerated shoots. Rooted plantlets survived well in a combination of pot mixture (1 sand: 1 soil: 1 FYM) and Vermiculite in the ratio of 1: 1.

ABSTRACT

Rubia cordifolia is an effective blood purifier in indigenous medicine. Habitat destruction and indescriminate harvesting is posing threat to its survival necessitating concerted efforts for conservation. The present study is an attempt to standardise in vitro multiplication protocol, a basic step for in vitro conservation, in Rubia cordifolia. Nodal explants cultured in MS basal medium supplemented with BA 1mg l ^-1 and IAA 0.2 mgl^-1 induced axillary bud break. The in vitro formed shoots developed roots on MS basal medium supplemented with IBA 1mg l ^-1. The plantlets were ready for plant out in 100 days of culture initiation and established well on planting out.

ABSTRACT

Vitex trifolia L. (Verbenaceae) is known for its medicinal property. Micropropagation can provide the opportunity to obtain large number of homogeneous plants and there are no reports of micropropagation of this species. So, we attempted for in vitro propagation of this species and described here is an efficient and rapid propagation method. This report describes in vitro shoot induction and plant regeneration from mature nodal explants of Vitex trifolia L. on Murashige and Skoog (MS) medium fortified with benzylaminopurine (BAP), kinetin (KN), thidiazuron (TDZ), adenine (ADE), and 2- isopentenyladenine (2 - iP) (0.25-10.0 mM). Multiple shoots differentiated directly without callus mediation within 3 weeks when explants were cultured on medium supplemented with cytokinins. The maximum number of shoots (9 shoots per explant) was developed on a medium supplemented with 5.0 mM BAP. Shoot cultures was established repeatedly by subculturing the original nodal explant on the same medium. Rooting of shoots was achieved on half strength MS medium supplemented with 0.5 mM naphthaleneacetic acid (NAA). Rooted plantlets were transferred to pots containing autoclaved soil and vermiculite mixture (1:1) that showed 90 % survival when transferred to outdoor. Plants transferred to the field have established themselves in the soil and are growing well. The regenerated plants did not show any immediately detectable phenotypic variation. Natural stands of Vitex trifolia are fast disappearing in India because of indiscriminate collection and over exploitation and the outlined procedure here offers a potential system for mass multiplication and conservation of Vitex trifolia.

ABSTRACT

Kattupadavalam (Trichosanthes cucumerina) is regarded as a laxative and blood purifier, considered beneficial in the treatment of skin diseases. Its entire supply being met from wild, unscrupulous exploitation has led to erosion of genetic resources of this crop. Hence it is imperative that in vitro trechniques are employed to conserve this valuable medicinal species and to evolve feasible protocols for large scale multiplication of this crop. Nodal cultures were established from two months old seedlings of Trichosanthes cucumerina and Murashige and Skoog (MS) medium, supplemented with BA @ 0.5 mg l^-1. Subculture of initiated shoots in the same medium at intervals of three weeks registered enhanced release of axillary buds producing 21 shoots from a single culture at the end of fifth subculture cycle. Elongated shoots were rooted in half strength MS medium, following a pulse treatment with IBA @ 1000 mg l^-1. A single stage root hardening process, wherein, rooted plantlets were subsequently transferred to half strength semisolid MS medium supplemented with 2 per cent sucrose, improved success rate. The rooted plantlets planted out in sterilized media and subjected to a hardening process in a phased manner, survived well.

ABSTRACT

Aquilaria agallocha Roxb. (Thymelaeceae), otherwise known as ‘Eagle wood’ is a large evergreen tree restricted to the eastern parts of India and Bhutan. The tree is valuable for its fragrant wood - ‘Agar wood’ which is tremendously used in the perfumery and incense stick industries. Lack of rapid natural regeneration and over exploitation has reduced its population drastically. Micropropagation offers a rapid means for clonal multiplication and conservation of this endangered species and attempts were made to standardize available protocol for in vitro multiplication. Media and conditions were standardized for culture establishment and propagule multiplication. Multiple shoots were induced in vitro from nodal segments of 10 yr old trees having enhanced axillary branching. Culture establishmnet was strongly influenced by the nature of the explant and the season of collecting the explants. Nodal segments collected during the period March- April from current season shoots showed a better response (90 %) in half strength Murashige and Skoog medium supplemented with 1.0 mg l^-1benzyl adenine and 0.1 mg l^-1kinetin. The rudimentary multiple shoots induced, proliferated in the basal media supplemented with benzyl adenine (0.1 mg l^-1), naphthalene acetic acid (0.5 mg l^-1) and coconut water (10%). This media also supported further multiplication in serial subculturing with a multiplication rate of 1:6. There exists no earlier report on in vitro multiplication of this tree species valued in perfumery.

ABSTRACT

Bacopa monnieri (L.) or Herpestis monniera, commonly known as brahmi, purifies the blood, and is useful in scabies, leucoderma, syphilis, diarrhoea and pyresis. As Brahmi is a major ingredient in several ayurvedic preparations, there is growing demand for steady supply of planting materials. Conventional propagation methods are less efficient than in vitro propagation. However, there are only a few reports on the in vitro propagation of this plant. Therefore, an attempt was made to evolve an improved protocol for the in vitro propagation of Bacopa. Shoot tips and nodal buds along with a portion of the stem excised from field grown disease free plants were taken as the explants. After three weeks of establishment on MS basal medium, the sterile cultures were sub-cultured on MS medium supplemented with different concentrations of BA (0.5,1.0 and 1.5mg l^-1). Rooting was induced on in vitro proliferated shoots by culturing on MS basal medium with NAA 1.0 mg l^-1 and IBA 0.5 mg l^-1. Treatments with BA 1.0 mg l^-1 and IAA 1.5 mg l^-1 recorded the maximum number of shoots (40.9 and 35.2, respectively). The height of the shoots was also significantly influenced by the concentrations of IAA (1.5 or 1.0 mg l^-1) in combination with BA (1.0 mg l^-1), recording 5.4 and 5.5 cm. The shoots with an average length of 3.0 to 4.0 cm were kept for rooting. NAA (1.0 mg l^-1) in combination with IBA (0.1, 0.5, 1.0 or 1.5 mg l^-1) produced significantly greater number of roots. The length of the roots was also greater in this treatment. The plantlets were transferred to pots in a greenhouse and later to the field with ninety per cent survival.

ABSTRACT

In vitro multiplication studies on two peach cvs namely Prabhat and Flordasun grown widely under agroclimatic conditions of Haryana were carried out using nodal and shoot tip explants. Amongst various media modification, M medium containing 1.5 mg^-1 BAP was found the best for shoot induction from both the explants. However, shoot tip showed better response in comparison to nodal explants. Shoot multiplication was highest on M⁷medium (MS+ 6 mg l^-1 BAP). Cultivar response did not differ significantly. Rooting was obtained on M medium containing 0.5 mg l^-1 NAA.

ABSTRACT

The present experiment was conducted to standardize the composition of nutrient media for callus regeneration. Shoot tip, internodal segment, leaf and hypocotyls of seedling grown under controlled condition were used as explants for callus induction. The study revealed that explants collected from younger seedlings showed greater proliferation of callus as compared to those collected from older one. The basal MS medium as well as WP medium fortified with different concentrations of auxins and cytokinins were tried. Initial better results of profuse callus growth with green colour and compact texture having regenerative capacity were obtained by using leaf explants. In all the treatments, the callus initiation occurred at the cut ends and then spread over the surface of the explant as a result of injury. The level of auxins presumable rises and quiescent cells were stimulated to divide rapidly producing callus. The leaf segment explant induced the callus after 9.50 days in medium WPM + NAA 2.5 mg/l with maximum callus growth (976.75 mg) with green, friable callus which was difficult to subculture. White and friable callus occurred within 12.00 days in MS + 2, 4 - D 5 mg/l. The green and compact callus having vigorous growth was observed in MS + NAA 2.5 mg/l within 12.75 days when subcultured on different regeneration media proved recalcitrant for shoot regeneration. On the regeneration medium the callus turned brown while some callus proliferates to produce white growth around it and dried later on.

ABSTRACT

In the studies on callus induction and somatic embryogenesis in acid lime (Citrus aurantifolia S.) var Sai-Sharbati, different ex-plants and different combinations of growth regulators (NAA, IBA and BA) in MS basal media were tried, the results indicated that cotyledonary segment was the best explant for callus induction among root tip hypocotyl stem, leaf, epicotyl stem and shoot tip explants tested of. The callus grown from epicotyl and hypocotyl stem explant was greenish white and compact. Whereas callus derived from cotylidonary segment and leaf segment was green and friable . Root tip and shoot tip did not exhibit callusing. The callus derived from epicotyl stem and hypocptyl stem explant responded shoot bud formation in MS medium supplemented with NAA 0.2mg/l + BA 0.5mg/l with or without ME 100mg/l. In somatic embryogenesis, full grown callus derived from epicotyl stem explant induced embryo formation within 28.33 days and more shoot bud (6.33) formation.

ABSTRACT

In vitro culture induced genetic variation constitutes an important source of variability for the improvement in ginger (Zingiber officinale Rosc.). Hence attempts were made to standardize indirect method of in vitro plantlet regeneration in ginger using various explants like sprouted bud, shoot tip, leaf and pseudo stem. Explants from two cultivars of ginger viz. Maran and Rio-de-janeiro were cultured in Murashige and Skoog (MS) medium supplemented with various levels of 2,4-D and in combination with different levels of BA. Cultures were incubated under two different culture conditions viz. light and dark. Callusing was observed in all the explants except mature leaf in MS medium supplemented with 2,4- D alone (1 - 2 mg l^-1) and in combination with BAP (0.5 - 3.0 mg l^-1). Shoot morphogenesis was achieved from one month old calli in MS medium supplemented with BAP (3.0 mg l^-1). Regenerated shoots were subcultured to medium of same composition for further shoot proliferation and rooting. Plantlets produced were hardened and planted out. From the present investigation, highly efficient protocol for indirect organogenesis in ginger could be developed and the response of two cultivars at various stages of morphogenesis could be studied in detail.

ABSTRACT

Aprotocol has been developed for the induction, maturation and germination of somatic embryos from leaf explants of Hevea brasiliensis (clone RRII 105). Leaf explants were cultured with their adaxial sides on modified Murashige and Skoog (MS) medium supplemented with different combinations of phytohormones such as 2,4-D & BA, NAA & BA as well as 2,4-D, BA and NAA. Compact calli could be developed from the cut ends of the explants on media containing 2,4-D (1.5 mg/l) and BA (1.0 mg/l) whereas pale yellow friable calli was obtained on media which contained NAA (0.2 mg/l) along with 2,4-D (1.2 mg/l) and BA (1.0 mg/l). The calli formed were detached from the explants and sub cultured for proliferation in medium containing reduced auxin (0.4 mg/l 2,4-D) and slightly increased level of sucrose (40 g/l). Proliferated calli were then sub cultured for embryo induction in modified MS medium supplemented with different auxins and cytokinins. Embryo induction could be achieved in modified MS medium containing BA (2.0 mg/l), GA (1.0 mg/l) and NAA (0.2 mg/l) and maturation occurred in WPM medium containing BA (0.3 mg/l), TDZ (0.5 mg/l) and GA (1.5mg/l). The cotyledonary stage embryos developed into plantlets on transfer to hormone free ½ MS medium. A distinct feature of this study is the induction of somatic embryogenesis from leaf explants of Hevea which has not been reported previously. This explant could be used further as an ideal target tissue for Agrobacterium mediated genetic transformation in Hevea.