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.