eChapters

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.

ABSTRACT

Immature zygotic embryos were isolated from field grown papaya fruits harvested at 90-105 d post-anthesis. The embryos were placed on half-strength Murashige Skoog (MS) medium supplemented with 2, 4-D (2.0 mg l^-1) and cultured under dark for a period of 2 months. The frequency of callus formation was around 66%. After 2 months, calli were transferred onto a half-strength MS basal medium and cultured for 3 months with frequent subculturing (once in 3 wk) on the same medium. The frequency of somatic embryo formation was around 36.6%. The somatic embryos on MS medium supplemented with BAP (0.4 mg l^-1) and NAA (0.02 mg l^-1) germinated and produced shoots. The emerging shoots of 2-3 cm height with 2-3 trilobed leaves were placed onto an MS basal liquid medium with IBA (1.0 mg l^-1) for root induction. The plants developed with shoots and roots were planted in a pot-mixture (sand:soil:FYM) containing VAM.

ABSTRACT

Shoot organogenesis from several explant types excised from in vitro grown plants of local elite brinjal (Solanum melongena L.) cv. Co2 was studied in response to the supplementation of thidiazuron (TDZ), BAP, IAA and NAA. Cotyledons and leaves were found to be the most responsive explants. High shoot bud induction rate (>50 shoot buds/explant) was achieved in the presence of 0.044 mg/l TDZ. The organogenic calli were subcultured in the same shoot bud induction medium for shoot development. Among different growth hormones, TDZ @0.044 mg/l induced more number of shoots per explant compared to BAP, IAA and NAA combinations. Efficient rooting was observed on half strength MS medium supplemented with 0.5 mg/l IAA. Higher percentage of rooting was achieved only when the well developed shoots were directly transferred to soil.

ABSTRACT

Papaya (Carica papaya L.) is an important crop in the tropics and subtropics for export of fruits and above all, for local consumption. Cultivation of papaya faces with problems due to its heterozygosity, dioecious nature and susceptibility to a large number of viral diseases. Crossing between two hermaphrodites will produce the highest ratio of fruit bearing female trees, which is of homozygous dominant andromonoecious types which are always letha l due to po st fertilization barriers leading to their abortion. The gynodioecious varieties of papaya have the M1M1 forms while selfing and hence for explant selection , gynodioecious varieties like Sunrise solo and CO7 were prefered Embryo culture is one of the earliest forms of in vitro culture applied to solve practical problems and is probably the tissue culture techniques that has proven of greatest value to breeders. Ovule culture is practised in a case when small (or) young embryos that abort at early stages of development and are difficult to isolate. This necessiates the development of a protocol for initiation, multiplication and regeneration of ovular callus of papaya.

ABSTRACT

The present experiment was conducted in order to develop a protocol for rapid in vitro propagation of pomegranate Cv. Mridula. Shoot tip and nodal segment explants selected from mature plant were cultured in MS medium supplemented with NAA and BAP in different concentration and combinations. To control phenolic browning, sub- culturing of cultures was under taken at an interval of first and third day after inoculation. The MS medium supplemented with NAA 0.4 mg/l + BAP 1.0 mg/l showed highest percentage of shoot differentiation with 77.77 per cent and 81.25 per cent response from shoot tip and nodal segment explant respectively. In the same media, earlier shoot initiation as well as more number of multiple shoots/culture were obtained. The shoots when elongated up to 2.5 - 3.0 cm length were transferred for rooting in the ½ strength MS media supplemented with auxins. There was no significant difference for root induction among the type of auxin used either NAA, IBA or both in combination. The concentration of auxins 0.5 mg/l was found to be optimum. Rooted plantlets after hardening in green house gave 49 per cent survival.

ABSTRACT

Direct shoot organogenesis from several explant types excised from in vitro grown plants of local elite tomato cv. PKM1 was studied in response to zeatin, BAP, IAA and NAA. Cotyledonary leaves were found to be the most responsive explants. High shoot bud induction rate (>25 shoot buds/cotyledonary explant) was obtained in the presence of 2 mg/l zeatin. The organogenic calli were subcultured onto the same shoot bud induction medium for shoot development. Among different growth regulators tested, zeatin @ 2 mg/ l induced more number of shoots per explant compared to BAP, IAA and NAA combinations. Rooting was efficiently induced on half strength MS basal medium supplemented with 0.5 mg/l IAA. The effects of various growth hormones on tomato regeneration on rooting were discussed.

ABSTRACT

Vanilla (Vanilla planifolia Andrews) is a high value spice crop having immense potential for cultivation in India as a source for natural vanillin. Though the crop was introduced way back in 1835, its cultivation gained momentum only in the recent past due to various reasons. Lack of sufficient good quality planting materials is one of the constraints for expanding the area under the crop. Conventionally vanilla is propagated by vine cuttings, but this method is inadequate to have large number of planting materials in a short span of time. So, micropropagation was attempted and proved to be highly successful. In the present study, performance of vanilla plants raised from tissue culture plantlets and vegetative cuttings was assessed on large-scale in planters’ field covering a total area of 111 ha in Kerala(68ha), Karnataka(28ha) and Tamilnadu(15ha) with the financial assistance of the Department of Biotechnology, New Delhi under the project entitled “Tissue culture Vanilla : Product Plan“. Disease free elite accessions of vanilla identified by the Indian Cardamom Research Institute (Spices Board), Myladumpara from its germplasm repository were used as mother plant source for tissue culture multiplication and also for raising the nursery for producing vegetative cuttings. The field planting for the demonstration plots was carried out during 1996-1999 on a cluster approach basis having 400 plants in each unit or plot of 0.25 ha. Accordingly a total of 444 evaluation plots were laid out under the project covering the aforesaid three states. Out of the 400 plants in a plot, 80% were nursery hardened grown up tissue culture plants and the balance( 20% )vegetative cuttings of comparable length. Observations on growth and yield attributes such as vine length, number of leaves per vine, internodal length, number of beans per vine, bean size, yield etc. revealed that performance of tissue culture plants is on par with that of conventional plants raised from vegetative cuttings of comparable length. It is also observed that if good management practices are adopted, tissue culture plants perform better at the full bearing stage of the plant. This proves that tissue culture plants of vanilla can be popularized as a cost effective and faster source of planting materials compared to conventional vegetative cuttings. As the demonstration plots were distributed under varied agroclimatic situations, the influence of different weather parameters on yield was also ascertained. Further, the project could convince the farmers about the comparable performance of tissue culture plants and that is quite evident from the ever increasing request for tissue culture vanilla plants received by the Spices Board.

ABSTRACT

Dissected shoot tips from healthy suckers of banana var. Meitei Hei (local variety) were inoculated on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of plant growth regulators viz., NAA and BAP. After 15 days of incubation 50% of the shoot tips turned green in the medium with 5.0 mgl^-1 BAP. A maximum of 85.7% of the explants produced phenolic exudates in MS + 5.0 mgl^-1 BAP. After one month the explants were subcultured in fresh medium with the respective concentrations and combinations of plant growth regulators. After 75 days of incubation an average of 2.20 microshoots were induced in 78.57% of the explants in MS + 5.0 mgl^-1 BAP. The regeneration of shoots, leaves and roots was more proportionate in MS + 0.1 mgl^-l NAA + 0.5 mgl^-1 BAP. Under the treatment the microshoots had an average height of 1.19 cm with 4.13 leaves and 2.40 roots. The multiple microshoots were separated and subcultured on MS + 0.1 mgl^-l NAA + 0.5 mgl^-1 BAP for proper development. After 120 days the completely regenerated plantlets were hardened under green house environment.

ABSTRACT

Vanilla planifolia Andrews - the high value, exotic spice crop has recently gained wide acceptance in the southern and northeastern states of the country. Being an orchid, germination of naked embryo is limited and this favours genetic uniformity. An attempt was made to induce genetic variability in Vanilla through biotechnology tools for its exploitation in crop improvement programmes. In vitro embryo culture and mutagenesis were applied for the purpose. Plantlets were multiplied, hardened, planted out and evaluated for morphological, biochemical and molecular markers. Great interclonal variability was observed in the in vitro derived plantlets. The intraclonal variability was very low in tissue culture derived regenerants. Isozyme and RAPD assay confirmed genotypic variation. Six superior clones were identified and distributed to farmers for further evaluation.

ABSTRACT

Astudy was undertaken to evaluate the performance of tissue culture derived plants vis a vis open pollinated seedlings of cardamom (Elettaria cardamomum Maton). A total of 102.5 ha (units) covering 50 ha in Kerala, 37.5 ha in Karnataka and 15 ha in Tamil Nadu was selected for the present study out of which each demonstration plot (unit) had an area of one hectare in which 80% of the plants are tissue cultured and 20% are open pollinated. Elite cardamom clones were subjected to in vitro multiplication and their respective open pollinated seedlings were compared. The planting was initiated in 1989 and completed with 1991 in all the zones identified. Plant growth characters such as number of tillers, number of bearing tillers and panicles per clump were recorded and compared. The yield was recorded in 1993-94 and 94-95 seasons after its stabilisation. Results indicated that tissue culture plants performed better compared to open pollinated seedlings with regard to growth attributes and yield irrespective of the seasons and zones. In Kerala region tissue culture plants exhibited a yield increase of 24.3 and 36% over open pollinated seedlings for the 1989 and 1990 plantings respectively. Similar trend was observed in Tamil Nadu for 1989 and 1990 planting where the percentage increase in yield became 130 and 21 respectively. In Kerala, the yield performance of tissue culture plants and open pollinated seedlings was on par during 1990 planting but in the subsequent year (1991) tissue culture plants showed 24% increase in yield compared to their respective open pollinated ones. Further, the study enabled to identify cardamom clones suited to in various zones of the cardamom tract. Accordingly 14 clones with a yield potential of over 750 kg/ha under moderate management were identified and this indicated that cardamom clones are highly location specific in their performance.

ABSTRACT

Recognizing the need to apply plant tissue culture tools for generating planting materials of important spice crops especially vanilla, the Spices Board started Biotech Production Unit at its head quarters building at Kochi with an annual production potential of 8 lakhs plantlets. Complete tissue culture protocols were developed and scaled up for large scale production of vanilla and other spices viz. large cardamom, small cardamom and tree spices. The laboratory is equipped with all modern facilities and is established in an area of about 2000 sft. Department of Biotechnology, Govt. of India also participated in the venture by sanctioning a project entitled “Regional centre for large scale production of tissue culture plantlets of vanilla and other spices” with a financial out lay of Rs 95 lakhs equally shared by DBT, New Delhi and Spices Board, Cochin.

At present there is a stock of over 3.00 lakh vanilla cultures in the lab and already 1.25 lakh plant lets were sent for hardening. The Spices Board, in coll aboration with “Kudumbashree”, a poverty eradication mission of Govt. of Kerala, extend short term training in tissue culture techniques to undergraduates from ‘Below Poverty Line’ (BPL) families and engage them as long term trainees in the Biotech Production Unit. The Board also coordinates and extends support to Kudumbashree members for, establishing and maintaining hardening facilities for TC plantlets thus providing means for empowering women from BPL families.

ABSTRACT

Jasmine is one of the economically most important flower crops cultivated for its fragrant flowers and valuable essential oil. In vitro studies to explore the possibility of regeneration of plantlets from different explants and to undertake biochemical analysis to detect the flavour compounds if present in the in vitro cultures were taken up with local varieties of Jasminum grandiflorum.

Among the different explants tried, leaf bits were found ideal for callus induction, followed by petals and shoot tips. MS medium with 2, 4-D at 1.25 ppm was the suitable medium for callus induction in leaf explants. Miller medium with NAA at 1.25 ppm and BAP at 1.25 ppm was the suitable medium for callus induction in petal explants collected from 18 days old buds of local varieties of Jasminum grandiflorum. Among the different media combinations tried, MS medium supplemented with 0.2 ppm NAA and 0.3 ppm GA₂ is the best medium for callus induction in shoot apex explants of 10mm size. Direct regeneration of plants was obtained in 10 per cent of shoot apex explants cultured in MS medium supplemented with 0.2 ppm GA₁ and 3 ppm BAP in 60 days. A maximum of 95 per cent embryogenicity is induced in leaf callus transferred to MS basal medium supplemented with 1.5 per cent sucrose from MS medium with 2, 4-D at 1.0 ppm.

The peroxidase assay indicated that embryogenic calli had six isoperoxidases while the non-embryogenic had only two. Biochemical analysis of the callus samples showed high Phenylalanine ammonia lyase and phenol indicating the initiation of phenolic pathway, which branches out and ultimately leads to the synthesis of flavour compounds in callus.

ABSTRACT

Donor-recipient protoplast fusion vis-à-vis cybridization is an effective technique to transfer plasmon-coded traits in cultivated tetraploid (2n=4x=48) potato (Solanum tuberosum L.) from its diploid (2n=2x=24) cross-incompatible wild relatives. However, the method, although attractive, is difficult in terms of not only inactivation of nuclear and plasmonic genomes of the participating donor and recipient protoplasts, but also post- fusion culture and regeneration of putative cybrids. Here, we report an improved method for donor-recipient protoplast fusion and subsequent post-fusion culture and regeneration of electrofused protoplasts in potato. In the present experiment, Solanum verrucosum Schlechtd. (2n=2x=24) accessions CPC 1339 (CGN No. 17774) and HAW 2246 (CGN No. 17766) containing sensitive [Tr^s] plasmon for tetrad-type cytoplasmic male sterility were used as donors and S. tuberosum subsp. tuberosum cvs Kufri Ashoka, Kufri Chipsona-1 and Professor Broekema as recipients. For nuclear inactivation, the in vitro-cultured donor plants were exposed to 1000 Gy of unfiltered γ-rays before protoplast preparation using a revised protoplast isolation medium (PIM) containing 2.0 % Cellulase ‘Onozuka’ RS and 0.25 % Macerozyme R-10. The recipient protoplasts were treated with antimetabolite iodoacetate (1.0 mM ) for 45 min at 10 °C for chondriome inactivation. The protoplasts were aligned in an amplitude of 300 Vcm^-1 for 0.5-1.0 s before being electrofused by one DC pulse of 600-800 Vcm^-1 for 40 μs using a Cell Electrofusion Processor FPH 01001 (Bioelectronics Corporation, Troy, Michigan). The fused protoplasts were immobilized in alginate-plates, and cultured on modified VKM medium containing 0.3 M glucose at about 400 mOsmol (pH 5.6). After 8-10 weeks, the developing protocalluses were dissociated from alginate-plates and cultured for regeneration on MS medium supplemented with varying concentrations of zeatin/ zeatin riboside and gibberellic acid. The protocalluses derived from electrofusion between donor S. verrucosum accession CPC 1339 and recipient S. tuberosum cv. Kufri Chipsona-1 could be successfully regenerated into plantlets. Morphological as well as molecular-genetic characterization of regenerated microplants, both for nuclear and chondriome genomes, confirmed the putative cybridity as well as established the extent and pattern of mitochondrial recombination following donor-recipient protoplast fusion. The results have been discussed in relation to donor-recipient protoplast fusion approach and its potential application in potato genetic improvement.

ABSTRACT

The investigation was carried out to develop protocol for micropropagation of acid lime (Citrus aurantifolia S) var. Sai-Sharbati. Shoot tip and nodal segment explants were obtained from axenically grown seedlings having 21 to 30 days age. Direct regeneration of shoots, roots and whole plant without intervention of callus was obtained from both the explants on MS basal media. In both the explants 100 per cent shoot proliferation was induced in nutrient medium MS + BA 0.25 mg/l + ME 200 mg/l. The shoot proliferation rate was more (5.0) in nodal segment as compare to the shoot tip explant (4.33). Regenerated shoots of more than 2.5 cm length with minimum of 4 leaves were transferred for rooting. Half strength MS medium supplemented with IBA 1.0 mg/l was best for root induction and root initiation within 15.66 days. More than 75 per cent plantlets were survived after hardening period of four weeks.

ABSTRACT

Immature seeds of Garcinia indica were excised from the green fruits and cultured on Woody Plant Medium (WPM) with a combination of auxins and cytokinins. Somatic embryos were obtained on the media supplemented with BAP (0.5-5.0 mg/l) alone or in combination with NAA 90.5 mg/l) with 80 per cent frequency within a period of 2-3 weeks. Subculture of primary embryos on maturation medium containing BAP (3.0 mg/l) supplemented with IAA (0.5-1.0 mg/l) and / or Kinetin (1.0 mg/l) gave rise to clusters of secondary somatic embryos. In the subsequent subculture on hormone free half strength WPM the embryo clusters germinated with repetitive secondary embryogenesis. About 70 per cent somatic embryos germinated into complete plantlets, which were successfully transferred to the green house conditions.

ABSTRACT

The totipotency of triploid endosperm tissue is exploited in many vegetatively propagated crops. Hevea brasiliensis, the most important commercial source of natural rubber, is a highly heterozygous perennial tree with a long breeding cycle. Conventional method of triploid production by crossing a diploid with an artificially induced tetraploid is a lengthy and laborious process. Hence in vitro regeneration of plants from endosperm tissue provides an easy and direct approach for the production of large number of triploids. Hevea fruits at different developmental stages were collected and sterilized using 0.1% HgCl₂ for 15 minutes. Seeds were dissected out and the embryos and inner integuments were removed. Endosperm is cut into small pieces and cultured over MS medium supplemented with various levels of sucrose, phytagel and growth regulators viz BA, Kin thidiazuron, 2,4-D, IBA, NAA, zeatin & GA. Organic supplements viz. yeast extract, casein hydrolysate, coconut milk and banana powder were used for callus and embryo induction. Optimum callus induction was obtained in media supplemented with 2,4-D (2.0 mg ^-1) and Kinetin (3.0 mg^-1). On further subculture of the primary calli to medium containing zeatin (1.0 mg ^-1), embryogenic calli could be obtained. Embryos were induced in MS medium supplemented with ABA (0.3 mg ^-1) , Kin (0.3 mg ^-1) and GA (0.5 mg ^-1). Maturation, germination and bipolar differentiation of the embryos were achieved in MS medium devoid of growth regulators. Root tips from the germinating embryos were subjected to cytological analysis and found to be triploids (3n=54). Regeneration from Hevea endosperm is being reported for the first time. The results indicates that like many other crops, Hevea endosperm responds well under in vitro conditions and endosperm culture could be utilized as a viable technique for triploid plant production.

ABSTRACT

Microbial contamination is perceived as the most serious threat faced by plant tissue culture research laboratories and commercial production units. A culture is often viewed as contaminated when obvious microbial growth incited by fungi, yeast or bacteria is noticed on the culture medium. On the other hand, there can be situations when microbes may be present in a covert or latent form with serious side effects on the cultures. The present investigations were taken up with the objectives of elucidating the cause of poor growth, lack of regeneration response or modification of rooting observed with visibly clean cultures of different horticultural crops. Apparently clean cultures of grape, banana, watermelon, eggplant, gerbera and chrysanthemum of recent origin or after long-term in vitro culturing were used in this study. Indexing the medium of such cultures using fungal/ yeast and bacteriological media brought out the covert association of one or more bacterial types with varying proportions of incidence in such cultures. Indexing of tissue revealed bacterial presence in different parts of in vitro plantlets. In some instances, bacteria showed endophytic colonization while being not detectable in the tissue culture medium subsequent to a disinfection or antibiotic treatment. Such bacterial association appeared to be the cause of in vitro decline observed with long-term micropropagated cultures, lack of reproducibility of tissue culture protocols, failure of in vitro rooting etc. besides being a threat to in vitro gene banks and germplasm exchange.

ABSTRACT

Techniques could be standardized for the induction, initiation, maturation and germination of somatic embryoids from the nucellar tissue isolated from 30-45 day old fruits of mono embryonic mango variety Neelum and poly embryonic variety Vellari Manga. The treatment half strength Murashige and Skoog basal medium containing 2,4-D 5.0 mgl^-1, GA3 5.0 mgl^-1, glutamine 400 mgl^-1, coconut water 200 mll^-1, activated charcoal 500 mgl^-1, sucrose 60 gl^-1and agar gl^-1was found to be the best in induction of embryogenic callus. The treatment 2,4-D 4.0 mgl^-1 + BA 1.0 mgl^-1+ GA3 5.0 mgl^-1in half strength MS basal medium supplemented with glutamine 400 mgl^-1, casein hydrolysate 500 mgl^-1, sucrose 60.0 gl^-1, coconut water 200 mll^-1and agar 6.0 gl^-1was the best in initiating somatic embryos from the induced nucellus of the three varieties. In the variety Neelum BA (8.0 and 16.0 mgl^-1) and kinetin (8.0 to 32.0 mgl^-1) were as effective as 2,4-D in inducing somatic embryogenesis. Polyembryonic variety Vellari Manga showed better response to induction treatments than the mono embryonic varieties. B5 major salts with MS minor salts in combination with 40 gl^-1sucrose, 10 gl^-1PVP, 200 mll^-1coconut water, 5.0 mgl^-1 ABA, 100 mgl^-1 casein hydrolysate and 6.0 gl^-1agar were the best suited for the maturation of the somatic embryos. The positive influence of cobalt chloride, casein hydrolysate and coconut water on the germination of embryoids of both mono and poly embryonic mango varieties could be well established. A basal medium consisting of B5 major salts and MS minor salts supported the highest percentage of germination of somatic embryoids in Neelum and Vellari Manga. However, abnormalities in germination were observed in most of the cases. Excellent germination of somatic embryos was observed in a liquid medium consisting of half strength B5 macro salts and full strength MS micro salts and supplemented with GA3 1.0 mgl^-1+ glutamine 400 mgl^-1 + sucrose 30gl^-1.

Waste is not actually waste but is the by-product of various activities of human life. The unclean handling of these wastes creates problem for public health departments and local bodies. The classification of waste depends on the agencies that classify these wastes according to the area of interest and method of disposal viz. medical field, agricultural field, local bodies, industries etc.

In the present paper, the emphasis is given to the classification based on agricultural activities which includes a) Agricultural marketing waste b) Meat and fish marketing waste.