eChapters

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.