eChapters

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.