IMPROVING GENE TRANSFER EFFICIENCY

A quick method of delivering foreign genes with high transformation effi ciency is required for the development of transgenic pulse crops. Agrobacterium-mediated gene transfer is the most common method of transformation of tropical grain legumes, although particle gun bombardment is also used to develop transgenic plants. Among the pulse crops, Vigna aconitifolia is the only pulse crop reported to produce regeneration of complete plants from isolated protoplasts (Eapen et al., 1987), which were exploited for genetic transformation using Agrobacterium (Eapen et al., 1987) and direct DNA transfer using PEG and electroporation (Kohler et al., 1987a, 1987b). However, these methods of gene transfer into protoplasts/cells cannot be applied to other pulse crops, where totipotency of protoplasts has not yet been demonstrated. In planta infection/electroporation of axillary meristems of pea, cowpea, and lentil for transformation, although reported by Chowrira et al. (1996, 1998), needs to be repeated by other laboratories, and also extended to other grains legumes. In lentil, vac- uum infi ltration with Agrobacterium was found to enhance transient gene expression (Mahmoudian et al., 2002). Although Agrobacterium is the most commonly used vector for transformation, it is worth trying other vectors such as Rhizobium (Broothaerts et al., 2005) for genetic transformation of pulse crops, since Rhizobium is a natural agent, which causes nodulation and nitrogen fi xation in leguminous plants. The development of quick transformation protocols—such as the imbibing of germinating seeds with Agrobacterium and the fl oral dip method as in the case of Arabidopsis (Clough and Bent, 1998)—is ideal and should be tested for the transformation of pulses. However, it is unlikely that the fl oral dip method of transformation will be successful in case of pulse crops due to an inability to reach the gametic cells because of the closed fl oral structure of legumes. Liu et al. (2005) developed a nontissue culture method for Phaseolus vulgaris using sonication and the vacuum infi ltration assisted Agrobacterium-mediated method for transformation.

Development of an in planta transformation method will overcome the tedious procedure of tis- sue culture and unwanted variation—somaclonal variation. Only one of the pulse crops, pigeon pea, is reported to have an in planta transformation (Rao et al., 2008). The protocol involves develop- ing whole plant transformants (to plants) directly from Agrobacterium-infected young seedlings. The apical and intercotyledonary regions are pricked with a needle, treated with Agrobacterium, and plants developed. The seeds produced were analyzed further and transgenics developed. This technique of gene transfer looks simple, but has to be repeated in other laboratories and with other pulse crops for the protocol to be of practical use. Microprojectile bombardment is also an effi cient method for developing transgenic plants, as has been demonstrated in the case of Vigna aconitifolia (Kamble et al., 2003), lentil (Gulati et al., 2002), and chickpea (Indurker et al., 2007). The develop- ment of transgenic pulse crops using Agrobacterium, particle gun bombardment, and other direct DNA transfer methods is shown in Tables 10.1, 10.2, and 10.3, respectively.

Despite several reports on the development of transgenic plants, transformation frequency in pulse crops has been very low. The selection of supervirulent strains of Agrobacterium with improved vir genes and the addition of phenolic compounds like acetosyringone, thiol compounds

TABLE 10.1

Selected Examples of Pulse Crop Transformation Using Agrobacterium tumefaciens

Plant Species Explant Used Fate of Explant Gene Reference

Cicer arietinum Leaf and stem Callus nptII Srinivasan et al. (1988, 1991) Embryos Plant uidA, nptII Fontana et al. (1993) Embryonic axis Plant cryIAc gene Kar et al. (1996,

1997)

Embryonic axis Plant nptII, uidA Krishnamurthy et al. (2000)

Embryonic axis and cotyledonary node

Plant uidA Sanyal et al. (2003) Plumule Plant bar Senthil et al. (2004) Half embryo with

one cotyledon

Plant _α-amylase

inhibitor

Sarmah et al. (2004) Embryo explant Plant aspartate kinase

gene

Tewari-Singh et al. (2004)

Embryo slice Plant uidA/nptII Polowick et al. (2004) Embryonic axis Plant cryIAc gene Sanyal et al. (2005) Embryos Plant α-amylase

inhibitor gene, nptII Ignacimuthu and Prakash (2006) Single cotyledon, half embryo explant

Plant Allium sativum

leaf agglutinin

Chakraborti et al. (2009)

Axillary meristems Plant PSCSF129A Bhatnagar-Mathur et al. (2009)

Cajanus cajan Shoot apices, cotyledonary node

Plant uidA, nptII Geetha et al. (1999) Embryonic axis Organogenesis,

and callus

nptII Lawrence and Koundal (2001) Embryonic axis and

cotyledonary nodes Organogenesis nptII hemagglutinin protein gene Satyavathi et al. (2003)

Leaf Plant uidA, nptII Dayal et al. (2003) Cotyledonary node Plant uidA Thu et al. (2003) Cotyledonary node Plant hptII, rice

chitinase

Kumar et al. (2004) Axillary bud of

germinating seed

Plant cryIAc gene Sharma et al. (2006) Plumular,

intercotyledonary region

Plant uidA, nptII Rao et al. (2008)

Lens culinaris Cotyledonary node, decapitated embryo

Plant nptII, uidA Sarkar et al. (2003) Cotyledonary node Plant nptII, uidA Celikkol et al. (2009)

Lupinus angustifolius L. Embryonal axis slices

Plants Sunfl ower seed albumin gene

Molvig et al. (1997) Shoot apieces Plant bar gene Pigeaire et al. (1997)

Phaseolus acutifolius Callus Plant uidA/nptII De Clerq et al. (2002)

TABLE 10.1 (continued)

Selected Examples of Pulse Crop Transformation Using Agrobacterium tumefaciens

Plant Species Explant Used Fate of Explant Gene Reference

Pisum sativum Shoot cultures Plant hptII Puonti-Kaerlas et al. (1990)

Immature embryo slices

Plant bar gene Schroeder et al. (1993) Immature embryo

slices

Plant bar gene Grant et al. (1995, 2003) Cotyledonary node Plant nptII, uidA Svabova et al. (2005)

Plant BAR, PGIP, VST1

Richter et al. (2007) Embryonic segments Plant uidA, bar gene Krejci et al. (2007)

Vicia faba Stem Plant uidA, lysC, methionine- rich sunfl ower 2S albumin

gene

Bottinger et al. (2001)

Embryo axes Plant SFAs gene, bar, lysC

Hanafy et al. (2005)

Vicia narbonensis Emryogenic callus Plant hptII Pickardt et al. (1991) Emryogenic callus Plant nptII, 2S

albumin gene,

PAT

Saalbach et al. (1994), Pickardt et al. (1995)

Vigna aconitifolia Protoplast Plant nptII Eapen et al. (1987) Leaf explant Plant uidA, nptII Malabadi and

Nataraja (2007)

Vigna angularis Epicotyl Plants hptII EL-Shemy et al. (2002)

Vigna mungo Cotyledonary nodes Plant uidA, nptII Saini et al. (2003) Embryonic axes Plants uidA, nptII Saini and Jaiwal

(2005) Shoot apex Plant uidA, nptII Saini and Jaiwal

(2007) Immature

cotyledonary node

Plants nptII, uidA, bar Murugananthum et al. (2007)

Vigna radiata Cotyledons Plants nptII, uidA Pal et al. (1991) Hypocotyl,

cotyledonary node, primary leaves

Callus, plants uidA, nptII Jaiwal et al. (2001) Leaf Plants nptII, uidA Tazeen and Mirza

(2004) Cotyledonary node Plant bar, α-amylase

inhibitor

Sonia et al. (2007)

Vigna sequidepalis Cotyledonary node Plants nptII, uidA Ignacimuthu (2000)

Vigna unguiculata Cotyledonary node Plants uidA, nptII Chaudhary et al. (2006)

Cotyledonary node Plants bar gene Popelka et al. (2006) Plant _α-amylase

inhibitor gene

such as l-cysteine (Olhoft and Somers, 2001; Olhoft et al., 2001) and the supplementation of the medium with osmotic agents may improve the transformation frequency in these important crop species. Some of the methods which can be used to improve the effi ciency of transformation include supplements of surfactants, controlling the density of Agrobacterium, altering the period and tem- perature of Agrobacterium cocultivation, vacuum infi ltration with Agrobacteria, sonication, subject- ing the explants to heat shock, and particle gun bombardment for inducing microwounds to help in

Agrobacterium-mediated gene transfer.