eChapters

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.