Vibha Dhawan

Establishment of genetic fidelity of In vitro-raised Lilium bulblets through rapd markers

SummaryRandom amplified polymorphic DNA (RAPD) markers were utilized for screening the clonal fidelity of in vitro-raised bulblets of Lilium sp. (Asiatic hybrids) produced through adventitious mode of propagation. The first set of 14 bulblets was randomly chosen from about 175 micropropagated bulblets regenerated from 1×1 cm2 bulbscale segments (explants) of cultivar ‘Gran Paradiso’ after four subcultures (after 6 mo.). The second set of 15 bulblets was selected again randomly after 12 subcultures (after one and a half years) when the bulblets were to be taken out for transplantation. Of the 20 primers used to screen the samples, only 14 primers gave clear reproducible bands. The 14 primers produced a total of 163 (an average of 11.6 bands per primer) scorable bands. Analysis of individual primers revealed the RAPD patterns produced were all shared by both the in vitro-raised bulblets (randomly selected after four and 12 subcultures) and the mother bulb. There was no variation observed within the tissue culture-raised progenies. Thus, our results show that adventitiously propagated in vitro bulblets of Asiatic hybrids of lilies are clonally uniform and stable.

A protocol for in vitro mass propagation of asiatic hybrids of lily through liquid stationary culture

SummaryA simple, rapid and cost-effective in vitro scheme has been proposed for mass propagating two cultivars of Asiatic lily hybrids. An average of seven bulblets was formed after 17 d when 1×1 cm2 bulb scale segments (explants) were cultured on Murashige and Skoog (MS) medium with 3% sucrose and 0.5 μM α-naphthaleneacetic acid (NAA). On MS medium containing 0.5 μM NAA and 6 or 9% sucrose, depending on the cultivar, large numbers of bulblets of increased size (3.5–5.0 cm in circumference) were formed under a 16/8 h photoperiod. A continuous system of mass propagation of bulblets was achieved through in vitro scale formation (secondary explants) on MS medium supplemented with 23 μM kinetin and 0.5 μM NAA, as well as scale proliferation on MS basal liquid stationary medium. Upon transplantation all bulblets sprouted, of which 40% flowered in the first season. Under ideal conditions, ca. 9.68×105 bulblets can be produced from a single scale segment in 1 yr by following the systematic propagation steps proposed here.

ISSR assay for ascertaining genetic fidelity of micropropagated plants of apple rootstock Merton 793

With the current trends in high density plantations of fruit trees, numerous clonal rootstocks of apple have been developed through various breeding programs. Among them, Merton 793 is the most popular in India because of the desirable traits of vigorous growth and resistance to woolly apple aphid and collar rot. The planting material of this rootstock cannot be multiplied at a desirable rate by means of conventional vegetative propagation methods, so micropropagation techniques are being explored to augment scarce planting material. Large number of plants can be produced in vitro under aseptic conditions, but there is always a danger of producing somaclonal variants by tissue culture technology. Thus, it is advisable to check the clonal fidelity of in vitro raised plants, especially of perennials prior to their field transplantation. The genetic stability of in vitro raised plants of apple rootstock Merton 793, multiplied through enhanced axillary bud proliferation up to 22 subculture passages, was tested by intersimple sequence repeat (ISSR) assay. Of 24 ISSR primers screened, 15 primers produced clear reproducible bands, resulting in a total of 134 distinct bands with an average of 8.9 bands per primer. Apple rootstock MM 111 and scion Jonathan, taken as outliers with tissue culture-raised progenies of Merton 793, ruled out the possibility that the invariant banding pattern occurred because of inefficiency of ISSR primers in detecting variations. The homogenous amplification profile observed for all the micropropagated plants compared to the donor plant confirmed the clonal fidelity of the tissue culture-raised Merton 793 plants. This suggests that axillary bud multiplication is the safest mode for multiplication of true-to-type plants. This is the first study that evaluates the applicability of ISSR markers in establishing clonal fidelity of tissue culture-raised apple plants.

Effect of nitrogen concentration on lipid productivity and fatty acid composition of Monoraphidium sp.

Green algae, Monoraphidium sp. T4X, was isolated locally, in New Delhi, India and identified as a potential source of biofuel. The study focuses on the effect of nutritional amendments and their uptake rates with respect to growth and change in fatty acid composition of the species. The lipid productivity and fatty acid profile were investigated and compared under six different nitrogen concentrations. Of the tested concentrations, cultures with nitrate concentration 0.36 g/l exhibited higher lipid productivity (0.18 g/l/day) with optimum content of all fatty acid compositions (SFA=37.22, MUFA=39.19, PUFA=23.60) with appropriate biodiesel properties. The right phase for harvesting microalgae was also investigated on the basis of the growth curve.

Large-scale production of Anogeissus pendula and A. Latifolia by micropropagation

SummarySuccess has been achieved in developing a complete protocol for mass propagation of Anogeissus pendula and A. latifolia, two important forest species found in India. Seeds cultured on plant growth regulator-free, semisolid Murashige and Skoog (MS) medium germinated within 5–6 wk and formed 4–6-cm long shoots. The shoots multiplied on MS+4.4 μM benzyladenine (BA)+5.7 μM indoleacetic acid (IAA) + casein hydrolysate (100 mgl−1) + ascorbic acid (50 mgl−1) + sucrose (3%) + agar (0.8%). A majority of the genotypes rooted with more than 90% efficiency when 5–6 cm individual shoots were cultured on 1/2MS (only major salts reduced to half strength)+2.3 μM IAA+2.5 μM indolebutyric acid (IBA) + sucrose (3%)+agar (0.8%) for 15 d. Those 10% (approx.) genotypes that did not root well on the above medium could be rooted with ease by increasing the concentration of IAA in the rooting media from 2.3 to 5.7 μM. The in vitro-raised plants were successfully transferred to the soil with a success rate of over 85%. Using this protocol, over 560 000 tissue-cultured plants of these two species have been produced and dispatched to various state forest departments for field trials and routine plantations.

Enhanced growth of micropropagated bulblets of Lilium sp. inoculated with arbuscular mycorrhizal fungi at different P fertility levels in an alfisol

Summary The effect of three arbuscular mycorrhizal (AM) inocula at four available levels of phosphorus (8.41, 12.53, 13.63 and 14.6 ppm) in non-disinfected soil was studied on the growth, flowering, P uptake and root colonization in micropropagated bulblets of Lilium sp. (Asiatic hybrid ‘Gran Paradiso’). The inoculated bulblets fared significantly better than the uninoculated ones in terms of all the growth variables, namely size, weight, shoot length, number of leaves and leaf area, and in P uptake. However, bulblets inoculated with different AM inocula had optimum growth at different P levels. Bulblets inoculated with indigenous mixed vesicular-arbuscular mycorrhiza species (VAM I) and Glomus intraradices isolate 2 (VAM III) showed the best growth and early flowering at available soil P of 13.6 ppm, whereas those inoculated with Glomus intraradices isolate 1 (VAM II) showed higher growth at 2.5 ppm available soil P. Amongst the three tested inocula, VAM I promoted maximum shoot length, bulblet size, and weight at 13.6 ppm P. The bulblets under this treatment also flowered earlier, nearly a month before the uninoculated control ones.

Development of a highly efficient micropropagation method for the Citrus rootstock 'Swingle' citrumelo [Poncirus trifoliata (L.) Raf. × C. paradisi McFaden].

This article describes a complete micropropagation protocol employing node segments from branches of adult trees of ‘Swingle’ citrumelo. Multiple shoots were induced in vitro through enhanced axillary branching. A multiplication rate of 3.79-fold every four weeks was achieved on Murashige and Skoogs media (1962) supplemented with cytokinins (6-benzyl amino purine and kinetin) with 4% sucrose and 0.4% agargel. The highest percent rooting was observed with a combination of indole-3-acetic acid and indole-3-butyric acid. The hardening survival rate of the rooted plantlets was 100% in a soil and agropeat combination. The micropropagated plantlets were tested for their genetic fidelity using seven inter simple sequence repeats primers.

Synergistic Effect of Polyamines on Bulblet Multiplication in Lillum sp (Asiatic Hybrids)

The in vitro bulblet multiplication of Lilium sp. (Asiatic hybrids) enhanced significantly by the application of polyamines than when cultured on medium containing high sucrose and auxin (NAA). Of the two polyamines tested, spermine was most effective. Maximum multiplication fold was 2.89 for cultivar ‘Gran Paradiso’ at 150 μM spermine and 2.78 fold for cultivar ‘sanciro’ at 50 μM. Putrescine also stimulated bulblet formation and multiplication to the tune of 2.56 and 2.61 fold at 50 μM as compared to control where it was 2.13 and 2.42 fold for cultivars ‘Gran Paradiso’ and ‘sanciro’, respectively. Though polyamines were effective in enhancing multiplication rates on an average of 26% in a year, growth of bulblets remained unaffected.

Sustainable Agriculture Practices for Food and Nutritional Security

The demand for increased food production and ecological threats for sustainable food production in years to come has resulted in relooking at the current agricultural practices. Globally, scientists are developing solutions for increasing crop productivity and developing nutritionally better crops that are more efficient in resource utilization. The challenge is not just restricted to more mouths to feed but also due to economic growth, the demand for food is increasing and so are the preferences for the kind of food. The availability of land for agricultural purposes is also on a decline because increase in population also demands additional infrastructure in terms of housing, land and industrial development. The threat of climate change is expected to have negative impact on agriculture especially in the developing part of the world as in the tropical region which is more densely populated. The impact might be more positive in temperate areas where temperatures are low and increase in temperature will actually help in agricultural growth. Unfortunately, water which is essential for plant growth is also becoming a scarce resource. All these demands for renewed thinking on agricultural practices being followed, becoming more innovative in terms of developing new technologies and developing policies for long-term sustainability for agriculture. The government policies are also shifting from providing more subsidies to favouring technologies that provide long-term ecological security. Issues related to some of the new technologies, such as biosafety concerns with transgenic crops and issues related to receding water table by some paddy varieties, are being seriously considered by the governments. This chapter discusses about issues related to sustainability of existing agriculture, lessons learnt from green revolution and possibility of new technologies so as to have sustainable ever-green revolution.

Cloning Forestry Species

Over-exploitation of Indian forests has led to progressive decline in the forest cover and its productivity. Since a large number of people depend on forest resources for their livelihoods and many more for meeting their energy needs it is imperative to enhance the productivity levels of our forests for a sustainable harvesting. Way back in 1999, the Department of Biotechnology, Government of India took the initiative of setting-up Tissue Culture Pilot Plants for micropropagation of various plant species. One of these facilities was established at TERI and so far about 12 million plants have been despatched out of which 3.7 million are of forest species alone. The field trials have clearly established clonal uniformity of tissue cultured plants and substantial increase in productivity levels.