ANS/PLSS 433:
Plant Cell, Tissue, and Organ Culture

Introduction: 

	The culture of plants in sterile culture has the potential to  
improve crops.  Two components are generation of variation and   
selection/propagation of variants. Combined with rDNA technology 
they allow plant transformation.

Plant Growth and Development: 

	Plants contain several apical meristems, in stems roots and buds. 
Meristems are undifferentiated cells which can give rise to all cell types. 
Outside the meristems cells do not divide merely expand. 

Plant Tissue Culture: 

	Tissue culture is the growth of plant parts (explants) in sterile 
culture on nutrient medium. Meristems are ideal tissues for culture because 
undifferentiated cells can divide and grow rapidly to form callus. Callus 
formation needs plant hormones in the media, usually an auxin a cytokinin 
and a gibberellin. Each plant species is different in its media requirements. 

Plant Cell Culture: 

	When callus or protoplasts are transferred to liquid media and 
agitated suspension cultures are derived. Such cells are subject to a high 
degree of genetic instability (somaclonal variation). Individual cells 
carrying novel mutations can be cloned and may be regenerated to plants.

Plant Organ Culture: 

	Apical meristems placed on media lacking plant hormones will 
develop seedling like shoots. Adding a cytokinin will produce axillary 
shoots. The shoot cluster can be divided, each shoot will reform another 
cluster (=micropropagation). Roots form on a media with auxin but lacking 
cytokinin.

Regeneration of Plants: 

	Roots can be induced to form on shoots or shoots from roots. Callus 
can be induced to form roots and shoots (organogenesis). Unfortunately 
monocots are the most difficult species to regenerate. They include rice, 
wheat and corn the 3/5 worlds important crops (woodfuel and soybean are the 
other 2). Their regeneration has been achieved by somatic embryogenesis 
where callus gives rise to embryos. These embryos can germinate to give 
plants. Embryogenesis is stimulated by removal of callus from auxin onto 
nitrogen rich media.

Anther and Pollen Culture: 

	Some pollen grains can be induced to form callus or embryoids. 
We can then regenerate true haploid plants.

Applications Non Breeding:

	1. Production of fine chemicals: 

		Plant materials are important sources of precursors and 
		products for pharmaceuticals, food, cosmetics and 
		agriculture. Cell culture might replace plant collection 
		but only if product value is more than $500/kg! Shikonin 
		production is the only example to date. The third world 
		contains most of the world potentially useful plants.

	2. Pathogen-free Plants: 

		Crop plants can be infected with a variety of microbial 
		pests which reduce yield and quality up to 90%. In many 
		cases even systemic infections can be eliminated my 
		meristem micropropagation. Taking a very small amount 
		or heat treating.

	3. Large Scale Propagation: 
		
		Some plants cannot be propagated vegetatively only from 
		seed. If like trees there is a long juvenility period 
		micropropagation can be invaluable eg. Oil palms, silver 
		Maple, Potato minitubers, Pacific Yew, orchard crops etc.

Applications of Plant Tissue Culture

Introduction

	Biotechnology applications of Plant tissue culture are manifold. 
One example of particular interest is the production of herbicide resistant
crops, particularly IT or IR corn (Imazethapyr Tolerant or Imazethapyr 
resistant).

The IT Method

	Cultures were initiated from corn hybrid A188xB73 because 
regeneration of plants is possible in that hybrid.  Immature embryos were 
used to initiate callus culture on agar with 1.5 mg/l auxin (24D) but no 
cytokinin.  A concentration of herbicide which inhibited cell growth 30% 
(50% of maximal inhibition was determined.That concentration was 0.37 µM 
imazethapyr.  After 10-30 d on that concentration callus showing good 
growth and embryogenic morphology (friable - brainlike) were transferred 
to fresh medium containing herbicide 3-5 times.  Herbicide concentration 
was increased 3 fold to 1 µM.  Resistant lines were regenerated into 
plants on agar with 0.1 mg/l auxin and 0.1 µM abscisic acid (no cytokinin), 
1 week in dark, 2 weeks in light.

Results

    	Eight IT (100-500 fold) plant lines were obtained. When tested for 
resistance to another herbicide, chlorsulfuron (a sulfonylurea) 4 lines 
were resistant to 1000 fold as callus. As seedlings resistances were lower 
(300xI and 100xC or 30xI respectively). Mutations are inherited as a 
single semidominant allele. All mutants produced novel herbicide resistant 
forms of the enzyme acetohydroxyacid synthase (AHAS) aka acetolactate 
synthase (ALS). Molecular genetic analysis in other plants suggest IT 
comes from Ser 653 to ASN (G 1958 to A). Sulfonylurea resistance comes 
from Pro 196 to Ser (C 591 to T). IT and S can come from Pro 196 with 
Trp 573 to Leu conversions.
 
Conclusion 

	Prolonged incubation on herbicide allows additional mutational 
events to improve resistance.

Other Herbicides

    	Similar approaches were attempted with Roundup resistance and 
Ignite resistance BUT no altered enzymes were obtained JUST highly 
increased gene expression. From Steve Padgetts seminar we know mutants are 
possible, but perhaps such alterations are highly deleterious to plant 
growth, remember the transgenic plants still had their wild type enzyme 
and the Roundup Ready mutant enzyme. If so the likelihood of weeds 
developing adequate resistance to Roundup or Ignite are very small.