S. S. Gosal

Biotechnology and Drought Tolerance

Abiotic stresses present a major challenge in our quest for sustainable food production as these may reduce the potential yields by 70% in crop plants. Of all abiotic stresses, drought is regarded as the most damaging. The complex nature of drought tolerance limits its management through conventional breeding methods. Innovative biotechnological approaches have enhanced our understanding of the processes underlying plant responses to drought at the molecular and whole plant levels. Hundreds of drought stress-induced genes have been identified and some of these have been cloned. Plant genetic engineering and molecular-marker approaches allow development of drought-tolerant germplasm. Transgenic plants carrying genes for abiotic stress tolerance are being developed for water-stress management. Structural genes (key enzymes for osmolyte biosynthesis, such as proline, glycinebetaine, mannitol and trehalose, redox proteins and detoxifying enzymes, stress-induced LEA proteins) and regulatory genes, including dehydration–responsive, element-binding (DREB) factors, Zinc finger proteins, and NAC transcription factor genes, are being used. Using Agrobacterium and particle gun methods, transgenics carrying different genes relating to drought tolerance have been developed in rice, wheat, maize, sugarcane, tobacco, Arabidopsis, groundnut, tomato, and potato. In general, the drought stress-tolerant transgenics are either under pot experiments or under contained field evaluation. Drought-tolerant genetically modified (GM) cotton and maize are under final field evaluations in the United States. Molecular markers are being used to identify drought-related quantitative trait loci (QTL) and their efficient transfer into commercially grown crop varieties of rice, wheat, maize, pearl millet, and barley.

In vitro selection at cellular level for red rot resistance in sugarcane (Saccharum sp.).

In the present study, in vitro selection technique using pathogen culture filtrate of Colletotrichum falcatum Went was employed with the aim to identify associations (if any), between selection at the cellular and plant level for red rot resistance in sugarcane (Saccharum sp.). Five to eight months old sugarcane calli of genotypes CoJ 88 and CoJ 64 were screened in vitro against pathogen culture filtrate for two selection cycles. Effect of pathogen culture filtrate on callus survival and/or proliferation was observed to be directly related to its concentration in the selection media. Calli survived and exhibited further proliferation at 5, 10 and 15% v/v pathogen culture filtrate concentrations whereas, at higher concentrations (20 and 25% v/v) proliferation was completely inhibited. Shoot regeneration percent was higher in calli selected on 5% pathogen culture filtrate concentration than those selected on 10 and 15% concentrations. In vivo screening of field transferred somaclones against two pathtypes (Cf 03 and Cf 08) showed considerable variation for red rot resistance. Somaclones regenerated from resistant and/or tolerant calli exhibited better resistance than the parental genotypes. The results indicated that in vitro selection for red rot resistance was effective and expressed when somaclones were screened in the field. This indicated a positive association between in vitro and in vivo methods of selection for disease resistance in sugarcane.

Role of Cysteine in Enhancing Androgenesis and Regeneration of Indica Rice (Oryza sativa L.)

The effects of amino acid cysteine to culture systems of microspore-derived callus induction as well as plantlet regeneration were studied. Isolated pollen along with anther walls of basmati cultivars, Pusa basmati 1, Basmati 370 and Basmati 386 were cultured in a medium based on N6 salts supplemented with or without cysteine following pollen embedment in agarose. The induction and regeneration medium with cysteine gave twice as effective androgenesis and plantlet regeneration in recalcitrant basmati rice cultivars as compared with medium lacking cysteine. Unlike the highly responsive model systems, most of the indica cultivars responded rather poorly in anther culture. So the study may accelerate the introgression of desirable genes into basmati rice using anther culture as a breeding tool. Response of microspores in androgenesis, plant regeneration and albinism was genotype specific. Regeneration of Indica rice varieties remains a limiting factor for researchers undertaking transformation experiments.

An efficient and reproducible method for regeneration of whole plants from mature seeds of a high yielding Indica rice (Oryza sativa L.) variety PAU 201.

Tissue culture is one of the tools necessary for genetic engineering and many other breeding programs. Moreover, selection of high regenerating rice varieties is a pre-requisite for success in rice biotechnology. In this report we established a reproducible plant regeneration system through somatic embryogenesis. The explants used for regeneration were embryogenic calli derived from mature seeds cultured on callus induction media. For callus induction mature seeds were cultured on MS medium containing 30 g/l sucrose combined with 560 mg/l proline and 1.5-3.5 mg/l 2,4-D and 0.5-1.5 mg/l Kin. For plant regeneration, embryogenic calli were transferred to MS medium containing 30 g/l sucrose, supplemented with 1.0-3.0 mg/l BAP, 0.5-1.5 mg/l Kin and 0.5-1.5 mg/l NAA. The highest frequency of callus induction (44.4%) was observed on the MS medium supplemented with 2.5 mg/l 2,4-D, 0.5 mg/l Kin, 560 mg/l proline and 30 g/l sucrose. The highest frequency of shoot regeneration (42.5%) was observed on the MS medium supplemented with 2.0 mg/l BAP, 0.5 mg/l NAA and 0.5 mg/l Kin. The plantlets were hardened and transferred to soil in earthen pots. The developed method was highly reproducible. The in vitro developed plants showed normal growth and flowering under glasshouse conditions.

Impact of cefotaxime on somatic embryogenesis and shoot regeneration in sugarcane

A cephalosporin antibiotic, cefotaxime (Omnatax™) promoted somatic embryogenesis and subsequent shoot regeneration in vitro from spindle in sugarcane irrespective of the genotypes as (CoJ 83, CoJ 88 and CoJ 64) culturered on MS medium with 2,4-D (2.5 mgl−1) and kinetin (0.5 mgl−1). Seven different concentrations of cefotaxime (100, 200, 300, 400, 500, 600 and 700 mgl−1) were tested to find the optimal concentration of cefotaxime for somatic embryogenesis from callus cultures. Among the three varieties, calli of variety CoJ 83 incubated on MS medium with 2,4-D (2.5 mgl−1) + kinetin (0.5 mgl−1) + cefotaxime (500 mgl−1) exhibited maximum somatic embryogenesis. To improve shoot regeneration, the callus was transferred to MS medium with BAP (0.5 mgl−1) + kinetin (0.5 mgl−1) in combination with different levels of cefotaxime. Highest frequency of shoot regeneration was observed in callus of CoJ 83 in the presence of 500 mgl−1 cefotaxime. The plantlets could be successfully hardened in polybags and transferred to soil, where they exhibited normal growth. Our results convincingly demonstrated that cefotaxime improves somatic embryogenesis from spindle and regeneration from embryogenic calli of sugarcane and hence can be strongly recommended for rapid and large scale multiplication of sugarcane.

Biotechnology and Crop Improvement

Plant biotechnology, a major component of agricultural biotechnology, deals with various aspects of plant tissue culture, genetic transformation, and molecular biology techniques. Tissue culture methods offer a rich scope for creation, conservation, and utilization of genetic variability for the improvement of field, fruit, vegetable, and forest crops, and medicinal/aromatic plants. Micropropagation technology ensures true to type, rapid and mass multiplication of plants that possesses special significance in vegetatively propagated plant species. This technology has witnessed a huge expansion globally, with an estimated global market of 15 billion US

Enhanced in vitro shoot multiplication and elongation in sugarcane using cefotaxime

/annum for tissue-culture products. Some basic techniques of tissue culture, such as anther/microspore culture, somaclonal variation, embryo culture, and somatic hybridization, are being exploited to generate useful genetic variability for obtaining incremental improvement in commercial cultivars. Production of secondary metabolites, such as food flavors, food colors, dyes, perfumes, drugs, and scented oils used in aromatherapy, through cell cultures and hairy root cultures, are leading examples of molecular farming. Cryopreservation of germplasm at the cell/tissue/organ levels, in liquid nitrogen at −196°C, is highly rewarding for establishing germplasm banks, especially for vegetatively propagated crops and rare, endangered plant species. During the past 15 years, remarkable achievements have been made in the production, characterization, field evaluation, and release of transgenic varieties/hybrids in several crops. Transgenic varieties/hybrids of maize, cotton, soybean, potato, tomato, and papaya are now being commercially grown on about 134 million hectares spread across 25 countries. Research in genomics allows high-resolution genetic analysis for physical mapping and positional gene cloning of useful genes for crop improvement. Molecular (DNA) markers help in precise characterization of germplasm, construction of saturated linkage maps, and DNA fingerprinting of crop varieties. Molecular markers are now increasingly being used for marker-assisted gene pyramiding and alien gene introgression. Current research, involving large-scale DNA sequencing, microarrays, and robotics, is heading towards gene revolution and nanobiotechnology.

Field performance of micropropagated plants and potential of seed cane for stalk yield and quality in sugarcane

Antibiotic cefotaxime has been found to be a growth promoting substance in sugarcane tissue culture. Young leaf segments (1–2 cm long) of three commercially important Indian sugarcane varieties viz., CoJ 83, CoJ 85 and Co 89003, regenerated into shoots and multiplied on semi-solid MS medium supplemented with 6-benzylaminopurine (BA, 0.2 mg/l) and kinetin (0.2 mg/l) for 2 cycles of 2 weeks each. Shoots (2 cm long) were separated and cultured on semi-solid half-strength MS medium supplemented with NAA (2 mg/l) and cefotaxime at various concentrations viz., 0, 250, 500 and 750 mg/l. Maximum shoot multiplication and elongation with respect to number of microtillers, shoot length and plantlet fresh weight in all the genotypes was obtained with cefotaxime used at the rate of 250 and 500 mg/l in the medium. Among the different varieties, on the basis of mean of 50 shoot cultures, number of microtillers per culture was highest in Co 89003 (7.50, 145.90% increase over control), whereas, shoot length and plantlet fresh weight were highest in CoJ 83, i.e. 10.30 cm (60.94% increase over control) and 360.45 mg (50.75% increase over control), respectively after 2 weeks of culturing with cephotaxime used at the rate of 500 mg/l in the medium. Statistical analysis of data revealed significant differences among varieties and media for different shoot multiplication and elongation parameters. Therefore, use of cefotaxime during tissue culture and genetic transformation of sugarcane can improve frequency of shoot multiplication and transformation, respectively.

Genetic engineering of Oryza sativa by particle bombardment

Two field experiments were conducted to ascertain the potential of micropropagation technique for faster production of seed cane by using tissue culture plants raised through apical meristem culture in first generation (TC 0) followed by clonal propagation through cane setts in next generation (TC1). About 18, 520 plants, produced from a single shoot through micropropagation, were required at row to row and plant to plant spacing of 90 and 60 cm, respectively as compared to 88 quintal of cane seed in conventional methods for planting in an area of one hectare. Multiplication ratio was 100–150 times using tissue culture plants as compared to 11–12 using conventional cane setts, leading to drastic reduction in seed cane requirement. The TC 1 exhibited superiority over vegetatively propagated conventional crop for millable canes and stalk yield by 17 and 10.4 per cent, respectively. Though the single cane weight and cane diameter (non-significantly) were slightly lesser in TC1 as compared to conventional crop, this did not distress its potential as seed crop. The incidence of Ratoon Stunting Disease (RSD) and Leaf Scald Disease (LSD) was very low in TC 1 crop as compared to conventional crop. The findings established the potential of tissue culture technique for the production of quality seed free of pests and pathogens in the existing varieties and rapid multiplication of newly released varieties for quick adoption by the growers.

Molecular Farming Using Transgenic Approaches

Genetic engineering of rice (Oryza sativa L. cv. Pusa basmati 1) using synthetic Cry1Ac gene has been achieved by “particle bombardment”. Scutellar tissues excised after 5 – 6 d from mature seeds cultured on induction medium were bombarded using gold particles coated with a mixture of Cry1Ac and marker genes on medium with osmoticum. Bombarded tissues were subjected to 30 mg dm−3 hygromycin selection for two cycles. The selected calli after GUS assay were transferred to shoot regeneration medium. Regenerated shoots were rooted and plantlets (T0) were grown to full maturity. Polymerase chain reaction (PCR) analysis of T0 plants using Cry1Ac specific primers revealed the presence of Cry1Ac gene in 65 % plants. Phenotypic assay, β-glucuronidase assay and PCR during T1 generation revealed the inheritance of the Cry1Ac and marker genes along with the native plant genes.